Nant cancer vaccine

ABSTRACT

Cancer is treated using coordinated treatment regimens that uses various compounds and compositions that drive a tumor from the escape phase of cancer immunoediting to the elimination and equilibrium phase of cancer immunoediting.

This application claims the benefit of priority to U.S. provisionalapplications having Ser. Nos. 62/357,324, filed on 30 Jun. 2016,62/371,665, filed on 5 Aug. 2016, 62/393,528, filed on 12 Sep. 2016,62/404,753, filed on 5 Oct. 2016, 62/463,037, filed on 24 Feb. 2017,62/474,034, filed on 20 Mar. 2017, and 62/473,207, filed on 17 Mar.2017.

FIELD OF THE INVENTION

The field of the invention is compositions and methods of cancertherapy, especially as it relates to cancer therapy in human.

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

More recently, the immune system was described as playing a dual role incancer as it can protect against cancer development by detecting andeliminating tumor cells, and as it can also promote cancer progressionby selecting for tumor cells that can escape immune destruction. Thisparadoxical role of the immune system in cancer is also referred to ascancer immunoediting (Cancer immunoediting: integrating immunity's rolesin cancer suppression and promotion. Science. 2011; 331:1565-70).Immunoediting is thought to include 3 phases, (1) elimination, in whichtumor cells are detected and eliminated by the immune system; (2)equilibrium, in which cancer cell killing is balanced by tumor growth;and (3) escape, in which tumor cell variants evade immune defenses andgrow rapidly.

Cancer cells harness various mechanisms to evade recognition anddestruction by immune cells (see e.g., The immune system and cancerevasion strategies: therapeutic concepts. J Intern Med. 2016;279:541-62). Cancer cells modulate in many cases the tumormicroenvironment (TME) through recruitment of regulatory T cells(Tregs), myeloid-derived suppressor cells (MDSCs), and immunosuppressivemacrophages (M2 macrophages). Cancer cells also evade the immune systemby down-regulating expression of certain MHC (major histocompatibilitycomplex) molecules, which are typically essential for T cells torecognize tumor-associated antigens (TAAs).

Traditional, molecularly uninformed treatment regimens of maximumtolerated dose (MTD) based chemotherapy, targeted therapy based oncancer marker signatures, and even monoclonal antibody therapy with highdose radiation impair the immune system, thereby generating tolerogeniccell death. Unfortunately, tolerogenic tumor cell death will enable theevasion of cancer immunosurveillance and facilitate the selection andescape of often multiple resistant, heterogenic clones with resultantmetastasis and poor long term outcomes in multiple tumor types. Thus,and contrary to their intent, the traditional regimens and currentstandards of care may inadvertently exacerbate and perpetuate the escapephase of cancer immunoediting, and support the immunosuppressive tumormicroenvironment, with poor long term outcomes in patients with cancer.Prior Art FIG. 1 exemplarily illustrates the three phases of cancerimmunoediting, depicting a path from healthy tissue to transformedcells, and the above noted three phases together with typicallyencountered factors and signaling molecules.

Indeed, it has now been realized that the long held assumption thatcancer cells grow in a linear fashion from a single clonally dominantmutant cell is largely incorrect, which has significant outcomeimplications both for the practice of high dose chemotherapy, as well asfor the administration of single agent targeted therapy. It is nowgenerally accepted that the vast majority of cancers arise and progressdue to numerous mutations in cancer cells, and that cancer is amulti-clonal disease. Moreover, and for the most part, each patient'scancer is unique in terms of the nature and number of mutations.Consequently, a paradoxical situation exists as it relates to thecurrent standard of care—that traditional MTD-based treatment regimensmay be eliciting a short-term response but at the same time driving thepatient's equilibrium phase into the escape phase by tilting the balanceof the tumor microenvironment into an immunosuppressive state. Indeed,the traditional regimens and current standards of care may inadvertentlyexacerbate and perpetuate the escape phase of tumor immunoediting, bysupporting the immunosuppressive tumor microenvironment resulting inpoor long-term outcomes in patients with cancer. This insight into thepotential cause for limited long-term remissions in most solid tumorsfollowing standard of care, requires a paradigm shift in the delivery ofMTD-based chemotherapy and single-agent targeted therapy.

The notion that formation of transformed (“cancer”) cells occurroutinely as part of the physiological process of regeneration, and thatclinical evidence of cancer is kept at bay during this dormancy phase(equilibrium) by the intact innate immune system of natural killer cells(elimination phase), as a normal physiological daily phenomenon in man,is intriguing. In this perspective, when the normal physiological stateis overwhelmed by mutations or by the immunosuppressive state of thetumor microenvironment, the escape phase ensues, with the resultantclinical evidence of cancer.

However, to this date no treatment regimen exists that attempts torevert tumor cells or tissue from the escape phase back to theequilibrium or even elimination phase. Therefore, while numeroustreatment compositions for cancer are known in the art, their use istypically limited to targeting specific defects in a tumor cell or toreduce checkpoint inhibition in a more general manner. Viewed from adifferent perspective, heretofore known cancer therapy is typicallyfocused on selected parameters of a tumor cell, in which recurrence isnearly a fait accompli where tumor heterogeneity is present.

Consequently, there is still a need to provide treatment compositionsand methods that address cancer immunoediting and that attempt to reverttumor cells or tissue from the escape phase back to the equilibrium oreven elimination phase in a patient-specific manner.

SUMMARY OF THE INVENTION

The inventive subject matter is drawn to various uses of compositionsand methods of cancer therapy in which various pharmaceuticalcompositions are administered to the patient to so revert tumor cells ortissue from the escape phase back to the equilibrium or even eliminationphase. Moreover, at least some of the pharmaceutical compositions arespecific to a patient and tumor in the patient, and will achieve in acoordinated fashion modulation of the tumor microenvironment to reduceimmune suppression and increase stress and damage signals in the tumor,induction and enhancement of innate and adaptive immune responses, andgeneration of immune memory.

In one aspect of the inventive subject matter, the inventors contemplatea method of treating a tumor that includes a step of reverting an escapephase of the tumor by administering at least a first pharmaceuticalcomposition that reduces immune suppression in a tumor microenvironment.In another step, the elimination phase is induced by administering atleast a second pharmaceutical composition that enhances an adaptiveimmune response and/or an innate immune response, and in a further stepthe equilibrium phase of the tumor is maintained by administering atleast a third pharmaceutical composition that biases the adaptive immuneresponse towards a T_(H)1 response.

In preferred aspects, the first pharmaceutical composition comprises adrug that is bound to albumin (e.g., nanoparticulate albumin). Wheredesirable, the albumin may further be coupled to an antibody or fragmentthereof to so further improve target specificity. Suitable drugs includeBendamustine, Bortezomib, Cabazitaxel, Chlorambucil, Cisplatin,Cyclophosphamide, Dasatinib, Docetaxel, Doxorubicin, Epirubicin,Erlotinib, Etoposide, Everolimus, Gefitinib, Idarubicin, Hydroxyurea,Imatinib, Lapatinib, Melphalan, Mitoxantrone, Nilotinib, Oxiplatin,Paclitaxel, Pazopanib, Pemetrexed, Rapamycin, Romidepsin, Sorafenib,Vemurafenib, Sunitinib, Teniposide, Vinblastine, Vinorelbine, andVincristine, while suitable antibodies or fragments thereof includeReopro, Kadcyla, Campath, Simulect, Avastin, Benlysta, Adcetris, Cimzia,Rbitux, Prolia, Zevalin, Tysabri, Gazyva, Arzerra, Xolair, Vectibix,Perjeta, Cyramza, Lucentis, Rittman, Bexar, Yondelis, and Herceptin.Alternatively, the antibody or fragment thereof may also bindspecifically to a component of a necrotic cell (e.g., nucleolin, DNA,etc.).

In still further contemplated aspects, suitable first pharmaceuticalcompositions may also comprise a drug that inhibits a T-reg cell, amyeloid derived suppressor cell, and/or a M2 macrophage. Thus, suitabledrugs include cisplatin, gemcitabine, 5-fluorouracil, cyclophosphamide,doxorubicin, temozolomide, docetaxel, paclitaxel, trabectedin, andRP-182 (see e.g., U.S. Pat. No. 9,492,499). Additionally, oralternatively, the first pharmaceutical composition may comprise avascular permeability enhancer (e.g., a portion of IL2).

With respect to suitable second pharmaceutical compositions it iscontemplated that such compositions may include a recombinant bacterialvaccine, a recombinant viral vaccine, or a recombinant yeast vaccine.Most typically, such vaccine is genetically engineered to express atleast one of a tumor associated antigen (e.g., MUC1, CEA, HER2,Brachyury, an oncogenic Ras mutant protein, etc.) and a patient andtumor specific neoepitope. Moreover, the second pharmaceuticalcomposition may also include a natural killer cell (e.g., an aNK cell, ahaNK cell, or a taNK cell), and/or an immune stimulatory cytokine (e.g.,IL-2, IL-15, IL-17, IL-21, IL-15 superagonist).

Contemplated third pharmaceutical compositions may comprise at least oneof a checkpoint inhibitor (e.g., PD-1 inhibitor or a CTLA4 inhibitor),an immune stimulatory cytokine (e.g., IL-2, IL-7, IL-15, IL-17, IL-21,IL-15, and superagonist versions thereof), a recombinant bacterialvaccine, a recombinant viral vaccine, and a recombinant yeast vaccine.

Additionally, contemplated methods may further include a step ofadministering low dose radiation to the tumor.

In another aspect of the inventive subject matter, the inventorscontemplate a method of treating a tumor. Such method will typicallyinclude a step of using omics information of a tumor and pathwayanalysis of the tumor to determine a chemotherapeutic treatment regimen,and a further step of administering the chemotherapeutic treatmentregimen at a low-dose metronomic schedule. In still another step, asecond treatment regimen is administered using at least onepharmaceutical agent that selectively delivers a drug to a tumormicroenvironment, and a third treatment regimen is administered using atleast one vaccine composition that is based on the omics information.Moreover, a fourth treatment regimen is administered that includes atleast one of a checkpoint inhibitor and an immune stimulatory cytokine.

Preferably the omics information comprises at least one of whole genomesequence information, exome sequence information, transcriptome sequenceinformation, and proteomics information, and/or the pathway analysis isa PARADIGM analysis. Notably, it should be appreciated that thechemotherapeutic treatment regimen is independent of an anatomicallocation of the tumor.

In further aspects of such methods, the at least one pharmaceuticalagent may comprise a drug that is bound to an albumin, wherein thealbumin is optionally a nanoparticulate albumin. Suitable drugs includeBendamustine, Bortezomib, Cabazitaxel, Chlorambucil, Cisplatin,Cyclophosphamide, Dasatinib, Docetaxel, Doxorubicin, Epirubicin,Erlotinib, Etoposide, Everolimus, Gefitinib, Idarubicin, Hydroxyurea,Imatinib, Lapatinib, Melphalan, Mitoxantrone, Nilotinib, Oxiplatin,Paclitaxel, Pazopanib, Pemetrexed, Rapamycin, Romidepsin, Sorafenib,Vemurafenib, Sunitinib, Teniposide, Vinblastine, Vinorelbine, andVincristine. Where desired, the agent may further comprise an antibodyor fragment thereof bound to the albumin, and preferred antibodies andfragment thereof include Reopro, Kadcyla, Campath, Simulect, Avastin,Benlysta, Adcetris, Cimzia, Rbitux, Prolia, Zevalin, Tysabri, Gazyva,Arzerra, Xolair, Vectibix, Perjeta, Cyramza, Lucentis, Rituxan, Bexar,Yondelis, and Herceptin.

Alternatively, or additionally, the at least one pharmaceutical agentmay also comprise a drug that inhibits at least one of a T-reg cell, amyeloid derived suppressor cell, and a M2 macrophage, and especiallypreferred drugs include cisplatin, gemcitabine, 5-fluorouracil,cyclophosphamide, doxorubicin, temozolomide, docetaxel, paclitaxel,trabectedin, and RP-182.

Most preferably, suitable vaccine compositions comprise a recombinantbacterial vaccine, a recombinant viral vaccine, or a recombinant yeastvaccine, which may be genetically engineered to express at least onepatient and tumor specific neoepitope.

With respect to checkpoint inhibitor it is preferred that the inhibitoris a PD-1 inhibitor or a CTLA4 inhibitor, and the immune stimulatorycytokine may be IL-2, IL-15, IL-17, IL-21, and/or an IL-15 superagonist.Additionally, contemplated methods may further comprise at least one ofadministration of a natural killer cell and low dose radiation.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an exemplary schematic prior art illustration of the threephases of cancer immunoediting.

FIG. 2 is an exemplary schematic illustration of a treatment accordingto the inventive subject matter.

FIG. 3 is an schematic illustration with exemplary compounds used inselected steps of a treatment according to the inventive subject matter.

FIG. 4 is an exemplary flow chart of a treatment according to theinventive subject matter.

FIG. 5 is a schematic illustration of mechanism(s) by which each agentis thought to impact the immune system, consequently leading toimmunogenic cell death of the tumor in the treatment of HNSCC.

FIG. 6 is a flow chart for administration of various pharmaceuticalcompositions during the induction phase in the treatment of HNSCC.

FIG. 7 is a flow chart for administration of various pharmaceuticalcompositions during the maintenance phase in the treatment of HNSCC.

FIG. 8 is a schematic illustration of a treatment regimen for HNSCCaccording to the inventive subject matter.

FIG. 9 is a schematic illustration of mechanism(s) by which each agentis thought to impact the immune system, consequently leading toimmunogenic cell death of the tumor in the treatment of MCC.

FIG. 10 is a flow chart for administration of various pharmaceuticalcompositions during the induction phase in the treatment of MCC.

FIG. 11 is a flow chart for administration of various pharmaceuticalcompositions during the maintenance phase in the treatment of MCC.

FIG. 12 is a schematic illustration of a treatment regimen for MCCaccording to the inventive subject matter.

FIG. 13 is a schematic illustration of mechanism(s) by which each agentis thought to impact the immune system, consequently leading toimmunogenic cell death of the tumor in the treatment of melanoma.

FIG. 14 is a flow chart for administration of various pharmaceuticalcompositions during the induction phase in the treatment of melanoma.

FIG. 15 is a flow chart for administration of various pharmaceuticalcompositions during the maintenance phase in the treatment of melanoma.

FIG. 16 is a schematic illustration of a treatment regimen for melanomaaccording to the inventive subject matter.

FIG. 17 is a schematic illustration of mechanism(s) by which each agentis thought to impact the immune system, consequently leading toimmunogenic cell death of the tumor in the treatment of NHL.

FIG. 18 is a flow chart for administration of various pharmaceuticalcompositions during the induction phase in the treatment of NHL.

FIG. 19 is a flow chart for administration of various pharmaceuticalcompositions during the maintenance phase in the treatment of NHL.

FIG. 20 is a schematic illustration of a treatment regimen for NHLaccording to the inventive subject matter.

FIG. 21 is a schematic illustration of mechanism(s) by which each agentis thought to impact the immune system, consequently leading toimmunogenic cell death of the tumor phase in the treatment of NSCLC.

FIG. 22 is a flow chart for administration of various pharmaceuticalcompositions during the induction phase in the treatment of NSCLC.

FIG. 23 is a flow chart for administration of various pharmaceuticalcompositions during the maintenance phase in the treatment of NSCLC.

FIG. 24 is a schematic illustration of a treatment regimen for NSCLCaccording to the inventive subject matter.

FIG. 25 is a schematic illustration of mechanism(s) by which each agentis thought to impact the immune system, consequently leading toimmunogenic cell death of the tumor in the treatment of PANC.

FIG. 26 is a flow chart for administration of various pharmaceuticalcompositions during the induction phase in the treatment of PANC.

FIG. 27 is a flow chart for administration of various pharmaceuticalcompositions during the maintenance phase in the treatment of PANC.

FIG. 28 is a schematic illustration of a treatment regimen for PANCaccording to the inventive subject matter.

DETAILED DESCRIPTION

Traditional molecularly uninformed treatment regimens using chemotherapyat the maximum tolerated dose (MTD), targeted therapy using kinaseinhibitors, agents that interfere with cell division, and antibodytherapy with high dose radiation typically impair the immune system andso generate tolerogenic cell death, which in turn enables the selectionand evasion of cancer immunosurveillance, and the escape of resistant,heterogenic clones with resultant metastasis and poor long termoutcomes. Thus, traditional regimens and current standards of care mayinadvertently perpetuate the escape phase of tumor immunoediting andsupport an immunosuppressive TME (tumor microenvironment).

A paradigm change in cancer care is required in which the treatment isbased on the biology of the tumor that is largely independent of theanatomy, the mechanism of cancer evolution, and that is specificallytailored to the genomic changes of the patient's tumor. The treatmentmethods and compositions presented herein represent such an approach.

According to the inventive subject matter, the inventors now discoveredthat cancer therapy can be targeted to maximize immunogenic cell death(ICD) while maintaining and augmenting the patients' antitumor adaptiveand innate responses to cancers. To that end, the treatment methods anduses of specific compounds and compositions presented herein takeadvantage of lower, metronomic doses of both cytotoxic chemotherapy andradiation therapy to so induce damage associated molecular patterns(DAMP) signals and tumor cell death while minimizing suppression of theimmune system. In addition, contemplated methods also include use ofvarious immunomodulatory agents, vaccines, checkpoint inhibitors,cell-based compositions, and fusion proteins to augment and stimulatethe patient's adaptive and innate immune responses. Notably, byovercoming the immunosuppressed TME, the elimination phase of cancer canbe reinstated through effector cells (e.g., mature dendritic cells, NKcells, cytotoxic T-cells, memory T-NK cells), that are preferablyactivated by combination therapy using fusion proteins, adenovirus andyeast vector vaccines, and natural killer cells. It should further beappreciated that such combinations will be targeted to the mutationalpatterns specific to the patients. Thus, off-target stimulation of animmune response is significantly reduced.

Most preferably, contemplated compounds and compositions areadministered in a temporal spatial orchestration of a combination ofimmunotherapeutic products to immunomodulate the tumor microenvironment,activate the innate adaptive immune system and to induce immunogeniccell death (ICD). More specifically, the inventors contemplate that suchapproach will result in coordinated effects, and especially in:

(1) Breaking the escape phase of cancer immune editing, preferably byovercoming the tumor immunosuppressed state. Such treatment ispreferably informed by tissue and/or liquid biopsies, executed withlow-dose metronomic chemotherapeutic agents capable of inhibiting T-Reg,MDSC's, and M2 Macrophages, and/or by inhibition of cytokines (e.g., TGF(β) which enhance immunosuppressive immune system;

(2) Inducing the elimination phase of cancer immune editing, preferablydone by up-regulating and/or induction of damaged associated molecularpatterns (DAMP) signals, up-regulating of tumor associated MHCrestricted antigens and stress receptors (NKG2D), up-regulating tumorspecific receptors such as PD-L1 and/or via low-dose radiation,administration of immunomodulatory drugs (IMiDs) and histone deacetylase(HDAC) agents, and/or activation of dendritic cells, natural killercells, cytotoxic T-cells, memory T and/or Natural Killer (NK) cellsthrough adenovirus, bacterial, and/or yeast vector vaccines, cytokinefusion protein administration, checkpoint inhibitors, and/or NK celltherapy infusion; and

(3) Reinstatement of the equilibrium phase of cancer immune editing,which can be achieved by maintaining T_(H)1 status of the patient'simmune system with vaccine boosters, cytokine fusion proteinmaintenance, and/or regular exogenous NK infusions.

Viewed from another perspective, the inventors contemplate that thetemporal spatial manner of contemplated treatments will recapture thenatural (pre-cancer) state of a patient's immune system by overcomingthe escape phase, reestablishing the elimination phase, and byaccomplishing long term maintenance through support of the equilibriumphase.

To that end, and among other contemplated options, preferred treatmentcomponents include (a) nanoparticle albumin bound (Nab) chemotherapycombinations to enter the tumor microenvironment (e.g., viatranscytosis) to overcome the tumor suppressor environment, (b) antigenproducing vaccine entities (e.g., recombinant adenovirus, bacteria,and/or yeast) that directly or indirectly deliver tumor associatedantigens and/or patient- and tumor-specific neoantigens to immunecompetent cells to activate immature dendritic cells in a patient andtumor specific manner to induce and/or enhance an adaptive immuneresponse, (c) natural killer cells, which may be endogenous (e.g., bystimulation with IL-15 or IL-15 superagonist) and/or exogenous (e.g.,genetically modified NK cells such as aNK, haNK, taNK cells) to induceand/or enhance an innate immune response, and (d) endogenous activatedmemory T- and/or NK-cells to sustain long term remission, preferablyactivated via vaccine, cell therapy, and fusion proteins (e.g.,genetically engineered fusion protein cytokine stimulators and/orcheckpoint inhibitors).

Therefore, and viewed from a mechanistic perspective, the inventorscontemplate that the temporal spatial orchestration of a combination ofimmunotherapeutic compounds and/or compositions will immunomodulate thetumor microenvironment, induce immunogenic cell death (ICD) and resultin long term sustainable remission of multiple tumor types with lowertoxicity and higher efficacy than current standards of care by (a)penetrating the tumor microenvironment to overcome the tumorimmunosuppressed state, which is preferably informed by tissue andliquid biopsies, with low-dose metronomic chemotherapeutic agentscapable of inducing immunogenic cell death (ICD), along with inhibitorsof one or more immunosuppressive cytokines; (b) up-regulating inductionof damaged associated molecular patterns (DAMP) signals, andup-regulating tumor associated MHC restricted antigens and stressreceptors (NKG2D) through low-dose radiation, IMiDs (immunomodulatorydrugs) and HDAC (histone deacetylating drugs) agents; (c) activatingdendritic cells, natural killer cells, cytotoxic T-cells, memory Tand/or NK cells through various cytokine fusion proteins, checkpointinhibitor administration, and NK cell therapy infusion; and (d)maintaining the equilibrium state through boost vaccines (e.g., antigenadenoviral, bacterial, and/or yeast vectors delivering tumor associatedand neoantigens), NK activating agents, and various immune stimulatingfusion proteins. Indeed, it should be appreciated that contemplatedmethods and uses take advantage of the tumor as a source of antigenicityand adjuvanticity.

Notably where a treatment approach according to the inventive subjectmatter is used, it should be recognized that most of the drugs in suchapproach are not primarily used in their traditional function (e.g., toblock a specific receptor or inhibit a specific enzyme) but that thedrug combinations are used in a concerted manner to modulate the immunebiology of the tumor and the immune system of the patient, therebyreverting the tumor from the escape phase to the elimination andequilibrium phase. In contrast, currently used combinations have so farfailed to make use of, or even appreciate modulation of cancerimmunoediting as a strategic approach in cancer therapy.

FIG. 2 exemplarily illustrates various aspects of the inventive subjectmatter. Here, as is schematically shown, while a tumor with multi-clonalcancer cells could be treated using standard of care, which will resultin tolerogenic cell death of a proportion of tumor cells, treatment willtypically result in a surviving cell fraction that represents cells thatare resistant to the standard of care and that have establish tumorsand/or metastases with a TME that is now immune suppressive andunresponsive to many treatment strategies. Moreover, it should be notedthat tolerogenic cell will generally not result in an immune stimulationas is typically encountered in ICD (immunogenic cell death—death of acell due to an immune response of a cancer patient against one or moreantigens of the tumor, typically via innate and adaptive immuneresponse).

In contrast, contemplated uses and methods are designed to first reduceor even revert immune suppression of the TME by use of compositions andcompounds that specifically or preferentially enter the TME as isfurther described in more detail below. In addition to the reduction orinhibition of immune suppression of the TME, contemplated methods anduses may further preferably comprise a low-dose metronomic chemotherapy.Such low-dose and metronomic chemotherapy advantageously allows thepatient's immune system to function to a degree that allows bothmounting of an innate and an adaptive immune response in atherapeutically effective manner.

Moreover, it is generally contemplated that such low-dose metronomicchemotherapy is informed by omics analysis and pathway analysis of thetumor of the patient. For example, omics analysis can identify specificmutations associated with a tumor as well as presence and expression ofneoepitopes specific to the patient and tumor. Thus, specific mutationscan be targeted with drugs know to treat such mutations (e.g., kinaseinhibitors for k-ras, etc.). In addition, thusly identified tumor andpatient specific mutations can also be used in immune therapy as isfurther described in more detail below. Preferably, omics analysis isperformed using a tumor and matched normal sample from the same patientas is exemplarily described in US20120059670 and US20120066001). Thus,it should be appreciated that omics analysis of a patient's tumor willnot only reveal druggable targets but also provide patient and tumorspecific neoepitope information that can be employed in immune therapy.

For example, patient- and tumor-specific neoantigens can be identifiedvia analyzing and comparing omics data from diseased tissue and healthytissue of a patient, (e.g., via whole genome sequencing and/or exomesequencing, etc.). Among identified mutations, it is generally preferredthat patient-specific neoantigens are further selected by filtering byat least one of mutation type, transcription strength, translationstrength, and a priori known molecular variations. Further details onidentification of patient-specific neoantigens and/or cancer-specific,patient-specific neoantigens are described in detail in theinternational patent application No. PCT/US16/56550.

Moreover, it is especially contemplated that the tumor-related antigenis a high-affinity binder to at least one MHC Class I sub-type or atleast one MHC Class II sub-type of an HLA-type of the patient, which maybe determined in silico using a de Bruijn graph approach as, forexample, described in WO 2017/035392, or using conventional methods(e.g., antibody-based) known in the art. The binding affinity of thehuman disease-related antigen is tested in silico to the determinedHLA-type. The preferred binding affinity can be measured by lowest KD,for example, less than 500 nM, or less than 250 nM, or less than 150 nM,or less than 50 nM, for example, using NetMHC. Most typically, theHLA-type determination includes at least three MHC-I sub-types (e.g.,HLA-A, HLA-B, HLA-C, etc.) and at least three MHC-II sub-types (e.g.,HLA-DP, HLA-DQ, HLA-DR, etc.), preferably with each subtype beingdetermined to at least 4-digit depth. It should be appreciated that suchapproach will not only identify specific neoantigens that are genuine tothe patient and tumor, but also those neoantigens that are most likelyto be presented on a cell and as such most likely to elicit an immuneresponse with therapeutic effect.

Of course, it should be appreciated that matching of the patient'sHLA-type to the patient- and cancer-specific neoantigen can be doneusing systems other than NetMHC, and suitable systems include NetMHC II,NetMHCpan, IEDB Analysis Resource (URL immuneepitope.org), RankPep,PREDEP, SVMHC, Epipredict, HLABinding, and others (see e.g., J ImmunolMethods 2011; 374:1-4). In calculating the highest affinity, it shouldbe noted that the collection of neoantigen sequences in which theposition of the altered amino acid is moved (supra) can be used.Alternatively, or additionally, modifications to the neoantigens may beimplemented by adding N- and/or C-terminal modifications to furtherincrease binding of the expressed neoantigen to the patient's HLA-type.Thus, neoantigens may be native as identified or further modified tobetter match a particular HLA-type.

Moreover, where desired, binding of corresponding wild type sequences(i.e., neoantigen sequence without amino acid change) can be calculatedto ensure high differential affinities. For example, especiallypreferred high differential affinities in MHC binding between theneoantigen and its corresponding wild type sequence are at least 2-fold,at least 5-fold, at least 10-fold, at least 100-fold, at least 500-fold,at least 1000-fold, etc.

In addition, the omics information (especially where the omicsinformation comprises whole genome sequencing or exome sequencing, RNAsequence and transcription data, and (preferably quantitative)proteomics information) can also be used to determine the status ofvarious cell signaling pathways. Such pathway information, andespecially in conjunction with mutational information, may revealfurther druggable targets within a cell that are independent fromanatomical features of the tumor (e.g., presence of HER2 signaling in anon-breast cancer). Particularly preferred pathway analyses that arebased on omics information include those described in WO 2011/139345, WO2013/062505, WO 2014/193982, WO 2014/059036, WO 2014/210611, WO2015/184439, and WO 2016/118527. Viewed from a different perspective,omics data in contemplated treatments and uses will be employed to both,inform generation of immune therapeutic compositions as well as informselection of chemotherapeutic drugs based on pathway information ratherthan tumor type and location. Therefore, suitable omics data includewhole genome sequencing data, exome sequencing data, RNA sequence andtranscription data, and proteomics data (e.g., quantitative proteomicsdata from mass spectroscopic analyses).

Use of genomics, transcriptomics, and proteomics data, especially inconjunction with pathway analysis of the obtained data allows foridentification of key altered cell signaling pathways, and with that anavenue to treatment that is agnostic to the anatomical type of tumor butsensitive to the functional alteration in signal transduction andassociated cellular events. This will not only allow for theidentification of drugs suitable for the treatment of the tumor thatwould otherwise not be considered, but also allow for modulation ofimmune parameters of the tumor. DNA, RNA, and protein signatures andassociated changes in signaling pathways can be identified, even beforetreatment begins. Indeed, non-assumptive stochastic analysis enablestreatment decisions that are unbiased to the traditionaltissue-by-tissue assignment of therapeutics or an a prior assumptionthat a few hundred DNA would be the drivers of the cancer.

With further respect to reduction or inhibition of immune suppression ofthe TME it is contemplated that the TME can be directly targeted withdrugs that preferentially accumulate in the TME. For example, directtargeting includes use inhibitors or T-regs (regulatory T cells), MDSC(myeloid derived suppressor cells), and/or M2 macrophages, use ofalbumin drug conjugates as further described below, and/or use of drugscoupled to antibodies or fragments thereof that bind to necrotic cells(e.g., nucleolin, histones, DNA, etc.)Indirect targeting will typicallyemploy permeability enhancing drugs that permeabilize the neovasculatureof the TME (e.g., IL-2 or PEP fragment thereof) to so allow facileaccess of drugs to the TME.

In still further contemplated aspects of reduction or inhibition ofimmune suppression of the TME, it is contemplated that the TME may alsobe subjected to stress conditions that induce the expression and displayof various stress signals, and especially NKG2D to so attract NK andother immune competent cells. For example, stress responses may beinduced using low dose radiation therapy (e.g., below 8Gy), hormonedeprivation, small molecule inhibitors, etc. Notably, where one or moreof the above approaches are taken, it is believed that at least some ofthe tumor cells will be subjected to exposure to various immunecompetent cells, and especially natural killer cells (which may be thepatient's own, or exogenous NK cells as described further below). Thus,addressing the TME may result in a first innate immune response.Advantageously, such innate immune response (e.g., via NK cells) willtrigger an immune cascade and stimulate adaptive immune response tocomponents of cells killed by the innate immune response. Therefore, itshould be appreciated that the treatments and uses certain compounds andcompositions can be employed to reduce or eliminate immune suppressionin the TME and as such can be used to block or revert the escape phaseof cancer immunoediting.

Upon reduction or reversal of immune suppression in the TME, orconcurrently with the reduction or reversal of immune suppression in theTME, the inventors contemplate that the elimination phase of the tumorcan be induced, preferably via one or more pharmaceutical compounds orcompositions that enhance at least one of an adaptive immune responseand an innate immune response. With respect to preferred induction ofadaptive immune response it is generally preferred that such response isgenerated by one or more vaccine compositions. For example, especiallypreferred vaccine compositions are formulated to generate an immuneresponse against tumor associated antigens (e.g., MUC-1, brachyury, CEA,HER2, etc.) and/or (preferably patient and tumor specific) tumorneoepitopes. In that context, it should be appreciated that the tumorneoepitopes used in the generation of the adaptive immune response willbe selected on the basis of the omics information as noted above.Advantageously, omics information for a specific patient is thereforeused for at least identification of a chemotherapeutic drug (preferablyvia pathway analysis using the omics data) and for identification ofsuitable neoepitopes to generate an immune therapeutic composition.

Among other suitable options, it is typically preferred that the immunetherapeutic composition is a cancer vaccine that is based on at leastone of a bacterial vaccine, a yeast vaccine, and an (adeno)viral vaccineas described in more detail below. It should be appreciated that thecancer vaccines are preferably recombinant entities that have expressedin the intracellular space one or more tumor associated antigens and/ortumor neoepitopes, or that the recombinant entity is a recombinant viralexpression vector that encodes. In further preferred aspects, it shouldalso be noted that the vaccine compositions may be administeredsequentially (e.g., first bacterial, then yeast, then viral), or thatonly one or two vaccine compositions are used (e.g., only adenoviral orbacterial vaccine). Of course, it should be appreciated that therecombinant protein(s) or nucleic acid(s) encoding the protein(s) may bethe same in all vaccine compositions, overlapping, or different.

With respect to the enhancement of the innate immune response in theelimination phase it is generally preferred that the innate immuneresponse may be from the patient's own immune system or via exogenousimmune competent cells. For example, where the patient's innate immuneresponse is enhanced, proliferation and activity of natural killer cellsand activated T-cells may be boosted using one or more immunestimulatory cytokines as discussed in more detail below. Alternatively,or additionally, the patient may also receive allogenic NK cells, andmost preferably activated NK cells (such as aNK cells, haNK cells, ortaNK cells) and/or recombinant T-cells with a chimeric T cell receptor.NK transfusion, and especially aNK and haNK transfusion advantageouslyamplify prior stress signals present on the tumor cells in the TME(typically induced by metronomic low dose chemo therapy, low doseradiation, and/or endocrine deprivation). Additionally, haNK cells maybe coupled via the high affinity CD16 receptor to one or more antibodiesthat bind tumor associated antigens or neoepitopes. As such, the innateimmune response may be specifically directed to a tumor cell. Theelimination phase may be further enhanced or supported by administrationof one or more cytokines, fusion proteins, and/or chemokines as isfurther discussed in more detail below.

Therefore, it should be appreciated that compounds and compositionsadministered to induce or enhance the elimination phase will beparticularly effective as the TME and the tumor were previouslyconditioned to have reduced or abrogated immune suppression and to haveadditional stress signals. Viewed from a different perspective, all oralmost all of the previously known treatments typically failed toexhibit therapeutic effect as such treatments were delivered oradministered to a TME that had maintained immune suppression. Incontrast, the presently contemplated methods and uses advantageouslyprecondition the tumor and the TME to so render treatments that inducethe elimination phase more effective. Where desired, the eliminationphase may be further supported by administration of one or more drugsthat inhibit T-regs, MDSCs, and/or M2 macrophages.

Upon induction of the elimination phase for a predetermined time orpredetermined treatment response, contemplated methods and uses willthen be directed to maintain the equilibrium phase. At this point,residual tumors, metastases, and tumor cells will have been largelyeliminated in a process that also stimulated an immune cascade (i.e., aprocess in which tumor cells attacked by immune competent cells (e.g.,NK cells, cytotoxic T cells) release immunogenic proteins of the tumor,leading to epitope spread and further immune response), leading toimmunogenic cells death and immune memory (e.g., memory T-cells, memoryB-cells, memory NK cells). To maintain the immune status of the patientand to further boost memory against the antigens that were present onthe tumor cells, the patient may receive checkpoint inhibitors, immunestimulatory cytokines, and/or further vaccine doses as described above.Such treatment and use of the above compounds and compositions will beeffective to bias the adaptive immune response and/or equilibrium phasetowards a T_(H)1 response (typically characterized by production ofInterferon-gamma, tumor necrosis factor alpha, and IL-2; in contrast, aT_(H)2 response is typically characterized by production of IL-4, IL-5,IL-6, IL-10, and IL-13). Maintenance using contemplated compounds andcompositions will maintain the equilibrium phase, support innate andadaptive immune response, and help generate memory NK, T- and B-cells.

Viewed form a different perspective, providing a treatment regimen thatreverts the escape phase of the tumor, that concurrently or morepreferably subsequently induces the elimination phase, and thatmaintains the equilibrium phase of the tumor can overcome immunesuppression and evasion of previously developed or established tumors.Thus, while contemplated methods and uses employ some of the samecompounds and compositions as traditional treatments, the coordinatedtreatment to achieve reversal of the escape phase to the eliminationphase and maintenance phase has neither been recognized nor appreciated.

FIG. 3 depicts schematically some of the compounds, compositions anduses that are contemplated. For example, the TME may be addressed usingabraxane (paclitaxel coupled to nanoparticulate albumin), variousantibody-drug conjugates that have an antibody portion that bindsspecifically to a component of a necrotic cell. For example, albumindrug conjugates may be used to exploit the gp60-mediated transcytosismechanism for albumin in the endothelium of the tumor microvasculature.Thus, various drug conjugates with albumin are contemplated in which adrug is non-covalently coupled to albumin (or nanoparticulate refoldedalbumin), and contemplated drugs include various cytotoxic drugs,antimetabolic drugs, alkylating agents, microtubulin affecting drugs,topoisomerase inhibitors, drugs that interferes with DNA repair, etc.Therefore, suitable drugs inclue Bendamustine, Bortezomib, Cabazitaxel,Chlorambucil, Cisplatin, Cyclophosphamide, Dasatinib, Docetaxel,Doxorubicin, Epirubicin, Erlotinib, Etoposide, Everolimus, Gefitinib,Idarubicin, Hydroxyurea, Imatinib, Lapatinib, Melphalan, Mitoxantrone,Nilotinib, Oxiplatin, Paclitaxel, Pazopanib, Pemetrexed, Rapamycin,Romidepsin, Sorafenib, Vemurafenib, Sunitinib, Teniposide, Vinblastine,Vinorelbine, and Vincristine. Such conjugates will advantageously beadministered in a low dose and metronomic fashion. Further contemplateddrugs for conjugation (or use without conjugation) to albumin includedrugs that inhibit suppressor cells in the TME, and especially T-regcells, myeloid derived suppressor cells, and/or M2 macrophages. Forexample such drugs include cisplatin, gemcitabine, 5-fluorouracil,cyclophosphamide, doxorubicin, temozolomide, docetaxel, paclitaxel,trabectedin, and RP-182 (see e.g., U.S. Pat. No. 9,492,499).

Likewise, where entry of a drug conjugate into the TME is mediated bythe FcRn receptor of the endothelium of the tumor microvasculature,various conjugates and chimeric proteins with the Fc portion of animmunoglobulin are contemplated. Thus, particularly contemplatedconjugates and chimeric proteins will include immune stimulatorycytokines (e.g., IL-2, IL15, etc.) and chemokines (e.g., CXCL14 CD40L,etc.). Alternatively, the TME may also be targeted in a morenon-specific manner by breaching the tumor microvasculature, typicallyusing a permeability enhancing peptide portion of IL-2 (PEP). Suchpermeability enhancers are preferably provided together with or prior toadministration of drugs that bind to necrotic tumor cells and/or drugsthat inhibit suppressor cells.

As is also schematically depicted in FIG. 3, immunogenicity of the tumorcells in the TME may be increased using one or more chemotherapeuticdrugs that are preferably selected on the omics and pathway analysis asnoted above. Such treatment is preferably performed at low dose and in ametronomic fashion to trigger overexpression or transcription of stresssignals. For example, it is generally preferred that such treatment willbe effective to affect at least one of protein expression, celldivision, and cell cycle, preferably to induce apoptosis or at least toinduce or increase the expression of stress-related genes (andespecially NKG2D ligands, DAMPsignals). It should be noted thatchemotherapeutic agents may advantageously stimulate both the innate andadaptive arms of the immune system by inducing an immunogenic type ofcell death in tumor cells resulting in the induction of specific damageassociated molecular pattern (DAMP) signals. These signals triggerphagocytosis of cell debris, promoting maturing of dendritic cells,activation of T- and NK cells, ultimately promoting anti-tumorresponses.

Thus, in contemplated aspects, treatment to increase immunogenicityand/or decrease immune suppression will include low dose treatment usingone or more of chemotherapeutic agents that target the TME. Mosttypically, the low-dose treatments will be at dosages that are equal orless than 70%, equal or less than 50%, equal or less than 40%, equal orless than 30%, equal or less than 20%, equal or less than 10%, or equalor less than 5% of the LD₅₀ or IC₅₀ for the chemotherapeutic agent.Viewed from a different perspective, low dose administration will be atdosages that are between 5-10%, or between 10-20%, or between 20-30%, orbetween 30-50%, or between 50-70% of a normally recommended dosage asindicated in the prescribing information for the drug. Additionally, andwhere desired, such low-dose regimen may be performed in a metronomicmanner as described, for example, in U.S. Pat. No. 7,758,891, U.S. Pat.No. 7,771,751, U.S. Pat. No. 7,780,984, U.S. Pat. No. 7,981,445, andU.S. Pat. No. 8,034,375.

In addition, contemplated treatments to target the TME to increaseimmunogenicity and/or decrease immune suppression may be accompanied byradiation therapy, and especially targeted stereotactic radiationtherapy at relatively low dosages (e.g., dosages that are between 5-10%,or between 10-20%, or between 20-30%, or between 30-50%, or between50-70% of a normally recommended dosage for radiation of the tumor). Totake advantage of expression and display or secretion of the stresssignals, it is generally preferred that low dose chemotherapy and/or lowdose radiation is followed within 12-36 by transfusion of NK cells(e.g., aNK cells, haNK cells, or taNK cells) to enhance an innate immuneresponse.

Therefore, it is contemplated that contemplated treatments and uses mayalso include transfusion of autologous or heterologous NK cells to apatient, and particularly NK cells that are genetically modified toexhibit less inhibition. For example, the genetically modified NK cellmay be a NK-92 derivative that is modified to have a reduced orabolished expression of at least one killer cell immunoglobulin-likereceptor (KIR), which will render such cells constitutively activated.Of course, it should be noted that one or more KIRs may be deleted orthat their expression may be suppressed (e.g., via miRNA, siRNA, etc.),including KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B,KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3,and KIR3DS1. Such modified cells may be prepared using protocols wellknown in the art. Alternatively, such cells may also be commerciallyobtained from NantKwest as aNK cells (activated natural killer cells).Such cells may then be further modified to express the co-stimulatorymolecules as further discussed below. In addition, contemplated NK cellssuitable for use herein also include those that have abolished orsilenced expression of NKG2A, which is an activating signal to Tregs andMDSCs.

Alternatively, the genetically engineered NK cell may also be an NK-92derivative that is modified to express a high-affinity Fcγ receptor(CD16-158V). Sequences for high-affinity variants of the Fcγ receptorare well known in the art, and all manners of generating and expressionare deemed suitable for use herein. Expression of such receptor isbelieved to allow specific targeting of tumor cells using antibodiesproduced by the patient in response to the treatment contemplatedherein, or supplied as therapeutic antibodies, where those antibodiesare specific to a patient's tumor cells (e.g., neoepitopes), aparticular tumor type (e.g., her2neu, PSA, PSMA, etc.), or antigensassociated with cancer (e.g., CEA-CAM). Advantageously, such cells maybe commercially obtained from NantKwest as haNK cells (high-affinitynatural killer cells) and may then be further modified (e.g., to expressco-stimulatory molecules).

In further aspects, genetically engineered NK cells may also begenetically engineered to express a chimeric T cell receptor. Inespecially preferred aspects, the chimeric T cell receptor will have anscFv portion or other ectodomain with binding specificity against atumor associated antigen, a tumor specific antigen, and/or a neoepitopeof the patient as determined by the omics analysis. As before, suchcells may be commercially obtained from NantKwest as taNK cells(‘target-activated natural killer cells’) and further modified asdesired. Where the cells have a chimeric T cell receptor engineered tohave affinity towards a cancer associated antigen or neoepitope, it iscontemplated that all known cancer associated antigens and neoepitopesare considered appropriate for use. For example, tumor associatedantigens include CEA, MUC-1, CYPB1, PSA, Her-2, PSA, brachyury, etc.

Moreover, it should be noted that the methods and uses contemplatedherein also include cell based treatments with cells other than (or inaddition to) NK cells. For example, suitable cell based treatmentsinclude T cell based treatments. Among other options, it is contemplatedthat one or more features associated with T cells (e.g., CD4+ T cells,CD8+ T cells, etc.) can be detected. More specifically, contemplatedomics analysis can identify specific neoepitopes (e.g., 8-mers to12-mers for MHC I, 12-mers to 25-mers for MHC II, etc.) that can be usedfor the identification of neoepitope reactive T cells bearing a specificT cell receptor against the neoepitopes/MHC protein complexes. Thus, themethod can include harvesting the neoepitope reactive T cells. Theharvested T cells can be grown or expanded (or reactivated whereexhausted) ex vivo in preparation for reintroduction to the patient.Alternatively, the T cell receptor genes in the harvested T cells can beisolated and transferred into viruses, or other adoptive cell therapiessystems (e.g., CAR-T, CAR-TANK, etc.). Beyond neoepitopes, the omicsanalyses can also provide one or more tumor associated antigens (TAAs).Therefore, one can also harvest T cells that have receptors that aresensitive to the TAAs identified from these analyses. These cells can begrown or cultured ex vivo and used in a similar therapeutic manner asdiscussed above. The T cells can be identified by producing syntheticversions of the peptides and bind them with commercially produced MHC orMHC-like proteins, then using these ex vivo complexes to bind to thetarget T cells. One should appreciated that the harvested T cells canincluded T cells that have been activated by the patient's immuneresponse to the disease, exhausted T cells, or other T cells that areresponsive to the discussed features.

Therefore, it should be noted that the above treatments will not onlytarget the TME to reduce immune suppression and increase immunogenicityof the tumor cells in the TME, but also initiate or support an innateimmune response. Advantageously, the innate immune response may befurther enhanced using tumor antigen specific antibodies that, whenbound to a tumor cell, trigger cytotoxic cell killing of NK cells.Notably, such antibodies can be targeted against known tumor associatedantigens (e.g., MUC-1, HER2, brachyury, CEA, etc.) but also againstpatient and tumor specific neoepitopes that were previously identifiedusing contemplated omics analyses. For example, preparation and use ofneoepitope specific antibodies are exemplarily described in WO2016/172722. Such antibody-mediated cell killing will also enhanceepitope spread (i.e., presentation of new tumor cell epitope viacytotoxic cell killing), which will in turn induce or enhance anadaptive immune response.

Still further, and with further respect to antibodies that bind to atumor cell antigen, it should be appreciated that such antibodies orfragments thereof may also be prepared as fusion proteins where thenon-antibody portion is an immune stimulatory cytokine, a chemokine, aco-stimulatory molecule, or a molecule that interferes with checkpointinhibition.

Viewed from a different perspective, tumor immunogenicity may begenerated or enhanced by tumor-specific binding of stimulating oranti-immune suppressive factors. Such treatment will advantageouslyinduce or enhance the elimination phase via at least one of innate andadaptive immune response.

With further reference to FIG. 3, adaptive immune response may also beinduced using one or more vaccine compositions that are tailored to thespecific patient's tumor via targeting tumor associated antigens and/ortumor neoepitopes. Where neoepitope vaccines are employed, it should berecognized that such neoepitopes advantageously are identified in theomics analyses as described above. There are various tumor vaccinecompositions known in the art, and all of them are deemed suitable foruse herein. However, especially preferred tumor vaccine compositionsinclude bacterial vaccine compositions in which the bacterium isgenetically engineered to express one or more tumor associated antigenand/or neoepitope. Most preferably, the recombinant bacterium isgenetically engineered such that it expresses endotoxins at a low levelthat is insufficient to induce a CD-14 mediated sepsis in the patient.One exemplary bacteria strain with modified lipopolysaccharides includesClearColi® BL21(DE3) electrocompetent cells. This bacteria strain isBL21 with a genotype F- ompT hsdSB (rB- mB-) gal dcm ion λ(DE3 [lacIlacUV5-T7 gene 1 indI sam7 nin5]) msbA148 ΔgutQΔkdsDΔlpxLΔlpxMΔpagPΔlpxPΔeptA. In this context, it should be appreciatedthat several specific deletion mutations (ΔgutQ ΔkdsD ΔlpxLΔlpxMΔpagPΔpxPΔeptΔ) encode the modification of LPS to Lipid IV_(A),while one additional compensating mutation (msbA148) enables the cellsto maintain viability in the presence of the LPS precursor lipid IVA.These mutations result in the deletion of the oligosaccharide chain fromthe LPS. Most typically, these bacteria are irradiated beforeadministration. Similarly, numerous yeast expression systems are deemedsuitable for use herein. However, especially preferred recombinant yeastsystems include those based on S. cerevisiae.

In still further preferred aspects of vaccine compositions, recombinantviruses are deemed suitable, and especially recombinant adenoviralsystems (such as Ad5 type) with reduced antigenicity as described inPCT/US16/65412, PCT/US17/17588, PCT/US17/23117, and WO 2016/164833. Suchviruses can, for example, be prepared in a method that includes one stepof identifying a cancer-related neoepitope of a patient, a further stepof determining binding of the neoepitope to an HLA-type of the patient,and determining an expression level of the neoepitope, a still furtherstep of selecting at least one co-stimulatory molecule, and a step ofgenetically modifying a virus to include a nucleic acid encoding the atleast one co-stimulatory molecule and the cancer-related neoepitope.With respect to the virus, it is generally referred that the virus is anadenovirus or a replication deficient virus. Moreover, it is furtherpreferred that the virus is non-immunogenic. Thus, especially preferredviruses include an adenovirus, and especially an Ad5 [E1⁻E2b⁻].

Cancer-related neoepitopes of the patient are preferably identified insilico by location-guided synchronous alignment of omics data of tumorand matched normal samples, and contemplated methods may furthercomprise a step of predicting the HLA type of the patient in silico.While not limiting to the inventive subject matter, it is preferred thatthe expression level of the neoepitope is at least 20% compared to amatched normal sample.

It is further contemplated that the recombinant entity (e.g., bacterium,yeast, virus) may also include one or more sequences that encode one ormore co-stimulatory molecule, including selected from the group of B7.1(CD80), B7.2 (CD86), CD3OL, CD40, CD40L, CD48, CD70, CD112, CD155,ICOS-L, 4-1BB, GITR-L, LIGHT, TIM3, TIM4, ICAM-1, and LFA3 (CD58).Moreover, the nucleic acid may further include a sequence encoding acytokine (e.g., IL-2, IL-7, IL-12, IL-15, an IL-15 superagonist(IL-15N72D), and/or an IL-15 superagonist/IL-15RαSushi-Fc fusioncomplex). Alternatively, or additionally, the nucleic acid further mayalso include a sequence encoding at least one component of a SMAC (e.g.,CD2, CD4, CD8, CD28, Lck, Fyn, LFA-1,CD43, and/or CD45 or theirrespective binding counterparts). Where desired, the nucleic acid mayadditionally comprise a sequence encoding an activator of a STINGpathway, such as a chimeric protein in which a transmembrane domain ofLMP1 of EBV is fused to a signaling domain of IPS-1. Such modificationsare thought to even further enhance development of an adaptive immuneresponse by providing additional signals for activation of the adaptiveimmune response.

Additionally, as also depicted in FIG. 3, the equilibrium phase may bemaintained or supported by administration of various cytokines andespecially IL-2 and IL-15, or a IL-15 superagonist, all of which may bepart of a fusion protein that has a binding portion that binds to atumor associated antigen, a necrotic cell component (e.g., nucleolin,DNA, a histone protein, etc.), or a patient and tumor specific antigen.Such compositions advantageously activate T-cells and NK cells at thetarget site of the tumor. Similarly, the equilibrium phase may bemaintained or supported by administration of various binders thatinterfere with checkpoint inhibition (e.g., PD-1 or PD-L1 binder), allof which may once more be part of a fusion protein that has a bindingportion that binds to a tumor associated antigen, a necrotic cellcomponent (e.g., nucleolin, DNA, a histone protein, etc.), or a patientand tumor specific antigen. In still further contemplated aspects ofenhancing the adaptive and/or innate immune response, administration ofhybrid proteins is contemplated in which the hybrid protein has aIL15/IL-15R-alpha component and an Fc component to stabilize the proteinand increase serum half-life time. For example, especially preferredhybrid proteins include IL-15-based immunostimulatory protein complexescomprising two protein subunits of a human IL-15 variant associated withhigh affinity to a dimeric human IL-15 receptor a (IL-15Rα) sushidomain/human IgG1 Fc fusion protein (J Immunol (2009) 183: 3598-3607).

Finally, as also illustrated in FIG. 3, contemplated methods and useswill include steps to maintain the equilibrium phase, typically byadministration of one or more inhibitors of suppressor cells such ascisplatin, gemcitabine, 5-fluorouracil, cyclophosphamide, doxorubicin,temozolomide, docetaxel, paclitaxel, trabectedin, and RP-182.Additionally, and where desired, checkpoint inhibitors can beadministered.

The spatiotemporal orchestration of treatment towards immunogenic celldeath is schematically illustrated in FIG. 4. Here, treatments and usesof contemplated compounds and compositions are shown as four elementsfrom a mechanistic point of view: Induction of ICD signals,consolidation of ICD signals, transplantation, and immune effectormaintenance.

As noted above, overcoming an immune suppressive TME will lay afoundation for later or concurrent treatments that are based on innateand adaptive immune responses. To that end, metronomic low dosechemotherapy may be given to a patient to enter the TME and toimmunomodulate the suppressor cells in the TME. Such phase can achievedin numerous manners, including the use of MDSC Inhibitors, T-Reginhibitors, M2 macrophage inhibitors, stimulation of M2 to M1transformation, modification of vascular permeability, administration ofVEGF and/or A2A R inhibitors, and even tissue oxygenation to thetypically hypoxic TME. Such treatment can be further augmented as notedabove with various compositions to increase the TME's immunogenicity.Induction of immunogenic signals can be achieved by chemotherapeutic,hormonal, and targeted therapy, as well as by epigenetic modulation(e.g., using histone deacetylases and other IMiDS such as DNMTinhibitors, HDAC inhibitors, SirT modulators, including azacitidine,decitabine, and vorinostat, etc.) to so increase the immunogenicity ofthe tumor cells. Depending on the type of treatment as discussed above,the primary tumor can become a source of vaccine antigens and immunestimulation (e.g., via release of DAMP signals or expression of stresssignals). Consolidation of the ICD signals is then performed viadendritic and T-cell conditioning using vaccine compositions asdiscussed above, typically along with immune stimulatory cytokinesand/or co-stimulatory signals. Treatments may also include theupregulation of tumor cell stress, receptor and/or antigen presentationwith radiation, typically at relatively low dose (e.g., <8Gy). Wheredesired or necessary, endothelial-to-mesenchymal transition may bemodulated, preferably by binding TGF-beta and and/or IL-10 toappropriate binding molecules. Transplantation preferably comprisesadministration of NK cells as already discussed above. Finally, immuneeffector maintenance can be achieved by administration ofimmunostimulatory cytokines, tumor vaccine boosters, and administrationof checkpoint inhibitors.

As will be readily appreciated, the methods and uses contemplated hereinwill be preferably accompanied by diagnostic tests to monitor treatmentefficacy, and suitable diagnostic test include radiology tests, biopsiesand attendant biochemical tests, omics analyses, and especially liquidbiopsy. Such monitoring will allow adjustment of one or more components,especially in view of newly discovered or recently eliminatedneoepitopes, newly discovered druggable targets and pathway activities,etc. In his context it should be noted that while it can take severalmonths for disease progression to show up on an imaging test, apan-omics approach, based on a patient's unique molecular profile andassociated proteomic signaling pathway signature, disease progressioncan be rapidly identified, allowing a change of the therapy to occur.

Circulating tumor RNA (ctRNA), and especially ctRNA with patient- andtumor-specific mutations, can be employed as a sensitive, selective, andquantitative marker for diagnosis and monitoring of treatment, and evenas discovery tool that allows repeated and non-invasive sampling of apatient. In most typical aspects, the ctRNA is isolated from a wholeblood sample that is processed under conditions that preserve cellularintegrity and stability of ctRNA and/or ctDNA. Notably, where ctRNA isisolated from a patient's biological fluid, miRNA (and other regulatoryRNA) can also be detected and/or quantified. Most typically, uponseparation of the ctRNA from non-nucleic acid components, circulatingnucleic acids may then be quantified, preferably using real timequantitative PCR.

Viewed from a different perspective, it should be appreciated thatvarious nucleic acids may be selected for detection and/or monitoring aparticular disease, disease stage, treatment response in a particularpatient, even before treatment has started. Advantageously, contemplatedcompositions and methods are independent of a priori known mutationsleading to or associated with a cancer. Still further, contemplatedmethods also allow for monitoring clonal tumor cell populations as wellas for prediction of treatment success with an immunomodulatory therapy(e.g., checkpoint inhibitors or cytokines), and especially withneoepitope-based treatments (e.g., using DNA plasmid vaccines and/orviral or yeast expression systems that express neoepitopes orpolytopes).

EXAMPLES

The following description provides exemplary protocols to treat cancerin a patient according to the inventive subject matter. It should beunderstood that while these protocols list specific compounds andcompositions alone or in combination, various alternative compounds andcompositions may be provided with the same or similar effect. Moreover,dosage and schedules may change according to patient age, stage ofcancer, and overall health condition.

Pharmaceutical agents and compositions: Unless otherwise noted herein,all of the compounds and compositions referred herein are known andcommercially available. The compounds that are not commerciallyavailable are characterized as listed below.

ALT-803: ALT-803 is an IL-15-based immunostimulatory protein complexcomprising two protein subunits of a human IL-15 variant associated withhigh affinity to a dimeric human IL-15 receptor a (IL-15Rα) sushidomain/human IgG1 Fc fusion protein (J Immunol (2009) 183: 3598-3607).The IL-15 variant is a 114 amino acid polypeptide comprising the maturehuman IL-15 cytokine sequence, with an asparagine to aspartatesubstitution at position 72 of helix C (N72D). The human IL-15Rα sushidomain/human IgG1 Fc fusion protein comprises the sushi domain of thehuman IL-15 receptor a subunit (IL-15Rα) (amino acids 1-65 of the maturehuman IL-15Rα protein) linked to the human IgG1 CH2-CH3 regioncontaining the Fc domain (232 amino acids). Except for the N72Dsubstitution, all of the protein sequences are human.

aNK: The aNK cell line is a human, IL-2-dependent NK cell line that wasestablished from the peripheral blood mononuclear cells (PBMCs) of a50-year-old male diagnosed with non-Hodgkin lymphoma (Leukemia 1994;8:652-8). aNK cells are characterized by the expression of CD56brightand CD2, in the absence of CD3, CD8, and CD16. A CD56bright/CD16neg/lowphenotype is typical for a minor subset of NK cells in peripheral blood,which have immunomodulatory functions as cytokine producers. Unlikenormal NK cells, aNK lacks expression of most killer cellimmunoglobulin-like receptors (KIR) (J Hematother Stem Cell Res 2001;10:369-83). Only KIR2DL4, a MR receptor with activating function andinhibitory potential that is expressed by all NK cells, was detected onthe surface of aNK. KIR2DL4 is considered to mediate inhibitory effectsthrough binding to the HLA allele G. The predominant pathway ofcytotoxic killing of aNK cells is through the perforin/esterase pathway;aNK expresses high levels of perforin and granzyme B )J Hematother StemCell Res 2001; 10:369-83).

aNK cells have a very broad cytotoxic range and are active against celllines derived from hematologic malignancies and solid tumors (Biol BloodMarrow Transplant 1996; 2:68-75). Safety assessments in severe combinedimmunodeficiency (SCID) mice showed no aNK treatment-related effects,such as acute toxicity or long-term carcinogenicity. Administration ofaNK cells to mice challenged with human leukemia cells or mouse modelsof human melanoma resulted in improved survival and suppression of tumorgrowth, including complete remissions in some mouse tumors.

haNK: The haNK cells are NK-92 [CD16.158V, ER IL-2] derivatives(high-affinity activated natural killer cell line, [haNK™ for Infusion])and cultured as a human, allogeneic, NK cell line that has beenengineered to produce endogenous, intracellularly retained IL-2 and toexpress CD16, the high-affinity (158V) Fc gamma receptor(FcγRIIIa/CD16a). Phenotypically, the haNK cell line is CD56+, CD3−, andCD16+.

The haNK cell line was developed by transfecting the parental aNK cellline with a bicistronic plasmid vector containing IL-2 and thehigh-affinity variant of the CD16 receptor (URL:https://nantkwest.com/technology/#hank). The plasmid contains anampicillin resistance cassette, and the promoter used for expression ofthe transgene is e longation factor 1 alpha (EF-1a) with an SV40polyadenylation sequence. The plasmid was made under transmissiblespongiform encephalopathies (TSE)-free production conditions andcontains some human origin sequences for CD16 and IL-2, neither of whichhave any transforming properties. haNK™ for Infusion has enhancedCD16-targeted ADCC capabilities as a result of the insertion of thehigh-affinity variant of the CD16 receptor. The haNK003 master cell bankwas derived from a monoclonal cell line.

Avelumab: Avelumab is a human monoclonal IgG₁ antibody that blocksinteraction between PD-L1 and its receptor, PD-1, while leaving intactinteractions between PD-L2 and PD-1 (see e.g., Lancet Oncol. 2016;17:1374-1385).

ETBX-011 (Ad5 [E1-, E2b-]-CEA(6D)): ETBX-011 is a Ad5 [E1-,E2b-]-CEA(6D) is an adenovirus vector vaccine in which the E1, E2b andE3 gene regions have been removed and replaced with a gene encoding CEAwith the CAP1-6D mutation (Cancer Immunol Immunother. 2015; 64:977-87;Cancer Immunol Immunother. 2013; 62:1293-301).

ETBX-021: ETBX-021 is a HER2-targeting adenovirus vector vaccinecomprising the Ad5 [E1-, E2b-] vector and a modified HER2 gene insert(Cancer gene therapy 2011; 18:326-335). The HER2 gene insert encodes atruncated human HER2 protein that comprises the extracellular domain andtransmembrane regions. The entire intracellular domain, containing thekinase domain that leads to oncogenic activity, is removed.

ETBX-051 (Ad5 [E1-, E2b-]-Brachyury): ETBX-051 is an Ad5-basedadenovirus vector vaccine that has been modified by the removal of theE1, E2b, and E3 gene regions and the insertion of a modified humanBrachyury gene. The modified Brachyury gene contains agonist epitopesdesigned to increase cytotoxic T lymphocyte (CTL) antitumor immuneresponses (see e.g., Oncotarget. 2015; 6:31344-59).

ETBX-061 (Ad5 [E1-, E2b-]-MUC1): ETBX-061 is an Ad5-based adenovirusvector vaccine that has been modified by the removal of the E1, E2b, andE3 gene regions and the insertion of a modified human MUC1 gene. Themodified MUC1 gene contains agonist epitopes designed to increase CTLantitumor immune responses (see e.g., Oncotarget. 2015; 6:31344-59).

GI-4000 (GI-4014, GI-4015, GI-4016, GI-4020): GI-4000 is 4 separateproducts from the GI-4000 series, GI-4014, GI-4015, GI-4016, GI-4020.Each of these is a recombinant, heat-inactivated S. cerevisiaeengineered to express a combination of 2-3 of the 6 mutated Rasoncoproteins. GI-4014, GI-4015, and GI-4016 products each contain twomutations at codon 61 (glutamine to arginine [Q61R], and glutamine toleucine [Q61L], plus one of three different mutations at codon 12(either glycine to valine [G12V], glycine to cysteine [G12C], or glycineto aspartate [G12D]). GI-4020 product contains two mutations at codon 61(glutamine to histidine [Q61H] and glutamine to leucine [Q61L]), plusone mutation at codon 12 (glycine to arginine [G12R]).

Thus, GI-4000 is manufactured as four individual products with thesubnames GI-4014, GI-4015, GI-4016, and GI-4020 depending on the mutatedRas oncoprotein the product is engineered to express. The biologicproduct is formulated in phosphate buffered saline (PBS) for injectionand vialed separately at a concentration of 20YU/mL (1YU=10⁷ yeastcells). Each single use 2 mL vial contains 1.2 mL of biologic product.Two vials of drug product will be used for each GI-4000 administrationvisit. The specific GI-4000 product containing the Ras mutation in thesubject's tumor will be used for treatment (GI-4014 for G12V, GI-4015for G12C, GI-4016 for G12D, GI-4020 for G12R or Q61H, and GI-4014,GI-4015, or GI-4016 for Q61L or Q61R). Two syringes of 0.5 mL will bedrawn from each vial, and 4 total injections will be administered for adose of 40YU at each dosing visit.

GI-6207: GI-6207 is a heat-killed, recombinant Saccharomyces cerevisiaeyeast-based vaccine engineered to express the full length humancarcinoembryonic antigen (CEA), with a modified gene coding sequence tocode for a single amino acid substitution (asparagine to aspartic acid)at the native protein amino acid position 610, which is designed toenhance immunogenicity. A plasmid vector containing the modified humanCEA gene is used to transfect the parental yeast strain (S. cerevisiaeW303—a haploid strain with known mutations from wild-type yeast) toproduce the final recombinant vaccine product (see e.g., Nat Med. 2001;7:625-9).

GI-6301: GI-6301 is a heat-killed, S. cerevisiae yeast-based vaccineexpressing the human Brachyury (hBrachyury) oncoprotein. The Brachyuryantigen is the full-length protein possessing an N-terminal MADEAP(Met-Ala-Asp-Glu-Ala-Pro) motif appended to the hBrachyury sequence topromote antigen accumulation within the vector and a C-terminalhexahistidine epitope tag for analysis by Western blotting (see e.g.,Cancer Immunol Res. 2015; 3:1248-56). Expression of the hBrachyuryprotein is controlled by a copper-inducible CUP1 promoter.

Head and Neck Squamous Cell Cancer (HNSCC):

Head and neck cancers collectively encompass a number of malignanttumors that involve the throat, larynx, nose, sinuses, and mouth. Anestimated 60,000 patients are diagnosed with head and neck cancerannually in the US and roughly half of all patients diagnosed with HNSCCdie of the disease. Despite various treatment options, there remains anurgent need to improve treatment outcome and overall survival.

In general, the overall goals of the HNSCC vaccine treatment presentedherein are to maximize ICD and augment and maintain the innate andadaptive immune responses against cancer cells. The rationale for theselection of agents is summarized in Table 1 in which i) denotes thattumor molecular profiling will determine whether ETBX-021 will beadministered; ii) denotes that tumor molecular profiling will determinewhether GI-4000 will be administered; iii) denotes that Capecitabine ismetabolized to 5-FU; iv) denotes that leucovorin potentiates theactivity of 5-FU; and v) denotes that either nivolumab or avelumab maybe administered.

Enhancing Mitigating Inducing and Conditioning Innate MaintainingImmunosuppression Coordinating Dendritic and Immune Immune Agent in theTME ICD Signals T Cells Responses Responses Non-Marketed productsALT-803 X X ETBX-011 X ETBX-021^(i)) X ETBX-051 X ETBX-061 XGI-4000^(ii)) X GI-6207 X GI-6301 X haNK cells X Approved productsBevacizumab X X Capecitabine^(iii)) X X Cetuximab X Cisplatin XCyclophosphamide X X 5-FU/leucovorin^(iv)) X X Fulvestrant XNab-paclitaxel X X Nivolumab/avelumab^(v)) X Omega-3-acid X ethyl estersSBRT X X

FIG. 5 exemplarily and schematically depicts the mechanism(s) by whicheach agent is thought to impact the immune system, consequently leadingto ICD. By combining agents that simultaneously (or sequentially) targetdistinct but complementary mechanisms that enable tumor growth, thetreatment regimen aims to maximize anticancer activity and prolong theduration of response to treatment.

To that end, contemplated HNSCC treatments combine low dose metronomicchemotherapy (LDMC), bevacizumab, cetuximab, cancer vaccine(s), low-doseradiation therapy, an IL-15 superagonist, NK cell therapy, and acheckpoint inhibitor. Such treatment regimen is thought to maximize ICDand augment and maintain the innate and adaptive immune responsesagainst cancer cells. More specifically, the treatment regimen is set upto interrupt the escape phase of immunoediting by (a) Mitigatingimmunosuppression in the TME. LDMC will be used to reduce the density ofTregs, MDSCs, and M2 macrophages contributing to immunosuppression inthe TME. Bevacizumab will be used to cause morphological changes in theTME to promote lymphocyte trafficking; (b) Inducing and coordinating ICDsignals. LDMC and low-dose radiation therapy will be used to increasethe antigenicity of tumor cells. Bevacizumab will be used to alter theTME, which allows for more efficient antigen-specific T-cell responsesand makes tumor cells more susceptible to ICD. Cetuximab and fulvestrantwill be used to enhance ADCC and cytotoxic T-cell activity; (c)Conditioning dendritic and T cells. Cancer vaccine(s) and an IL-15superagonist will be used to enhance tumor-specific cytotoxic T-cellresponses; (d) Enhancing innate immune responses. NK cell therapy willbe used to augment the innate immune system. An IL-15 superagonist willbe used to enhance the activity of endogenous and introduced NK cells.Low-dose radiation therapy will be used to stimulate the activity of NKcells; and (e) Maintaining immune responses. A checkpoint inhibitor willbe used to promote long-term anticancer immune responses.

The HNSCC vaccine treatment will be conducted in 2 phases: an inductionphase and a maintenance phase. The purpose of the induction phase is tostimulate immune responses against tumor cells and mitigateimmunosuppression in the TME. The purpose of the maintenance phase is tosustain ongoing immune system activity against tumor cells, creatingdurable treatment responses. Exemplary use and timing of administrationof contemplated compounds and compositions for the induction phase andthe maintenance phase are shown in FIG. 6 and FIG. 7, respectively.Therefore, the following agents and compositions are preferably used forthe induction and maintenance phases:

1. ALT-803, recombinant human super agonist IL-15 complex (also known asIL 15N72D:IL-15RαSu/IgG1 Fc complex); 2. ETBX-011 (Ad5 [E1-, E2b-]-CEA);3. ETBX-021 (Ad5 [E1-, E2b-]-HER2); 4. ETBX-051 (Ad5 [E1-,E2b-]-Brachyury); 5. ETBX-061 (Ad5 [E1-, E2b-]-MUC1); 6. GI-4000 (Rasyeast vaccine); 7. GI-6207 (CEA yeast vaccine); 8. GI-6301 (Brachyuryyeast vaccine); 9. haNK™, NK-92 [CD16.158V, ER IL-2], Suspension for IVInfusion (haNK™ for Infusion); 10. Avelumab (BAVENCIO® injection, for IVuse); 11. Bevacizumab (AVASTIN® solution for IV infusion); 12.Capecitabine (XELODA® tablets, for oral use); 13. Cetuximab (ERBITUX®injection, for IV infusion); 14. Cisplatin (CISplatin injection); 15.Cyclophosphamide (CYCLOPHOSPHAMIDE Capsules, for oral use); 16. 5-FU(Fluorouracil Injection, for IV use only); 17. Fulvestrant (FASLODEX®for injection); 18. Leucovorin (LEUCOVORIN Calcium for Injection, for IVor IM use); 19. Nab-paclitaxel (ABRAXANE® for Injectable Suspension[paclitaxel protein-bound particles for injectable suspension][albumin-bound]); 20. Nivolumab (OPDIVO® injection, for IV use); 21.Omega-3-acid ethyl esters (Lovaza capsules, for oral use); and 22.stereotactic body radiotherapy (SBRT).

More specifically, an exemplary treatment protocol for HNSCC willtypically include the following steps, phases, compounds andcompositions:

Tumors will be assessed at screening, and tumor response will beassessed every 8 weeks during the induction phase, and every 3 monthsduring the maintenance phase by computed tomography (CT), magneticresonance imaging (MRI), or positron emission tomography (PET)-CT oftarget and non-target lesions in accordance with Response EvaluationCriteria in Solid Tumors (RECIST) Version 1.1 and immune-relatedresponse criteria (irRC).

Prospective Tumor Molecular Profiling: Prospective tumor molecularprofiling will be conducted to inform HER2 expression and Ras mutationalstatus and will be used to determine whether ETBX-021 and GI-4000 willbe administered. All subjects will receive ETBX-011, ETBX-051, ETBX-061,GI-6207, and GI-6300 regardless of their tumor molecular profile.Prospective tumor molecular profiling will be performed on FFPE tumortissue and whole blood (subject-matched normal comparator against thetumor tissue) collected at screening. Subjects will receive ETBX-021 iftheir tumor overexpresses HER2 (≥750 attomole/μg of tumor tissue, asdetermined by quantitative proteomics with mass spectrometry). Subjectswill receive GI-4000 if their tumor is positive for specific Rasmutations, as determined by whole genome sequencing. As noted above,GI-4000 is 4 separate products from the GI-4000 series (GI-4014,GI-4015, GI-4016, and GI-4020); each of these expresses a combination ofmutated Ras oncoproteins. The specific Ras mutation will determine whichGI-4000 product will be used for treatment (GI-4014 for G12V, GI-4015for G12C, GI-4016 for G12D, GI-4020 for G12R or Q61H, and GI-4014,GI-4015, or GI-4016 for Q61L or Q61R).

Induction Phase: The induction phase will comprise repeated 2-weekcycles for a maximum treatment period of 1 year. The treatment regimenof omega-3-acid ethyl esters, cyclophosphamide, cisplatin, 5FU/leucovorin, nab-paclitaxel, bevacizumab, ALT-803, haNK cells,Ad5-based vaccines (ETBX-011, ETBX-021, ETBX-051, and ETBX-061),yeast-based vaccines (GI-4000, GI-6207, and GI-6301), nivolumab oravelumab, fulvestrant, cetuximab, and radiation therapy will be repeatedevery 2 weeks. Concurrent SBRT will be given during the first four2-week cycles. Radiation will be administered to all feasible tumorsites using SBRT. Specifically, an exemplary induction phase of thetreatment will be conducted in accordance with the following dosingregimen:

Daily:

Omega-3-acid ethyl esters (by mouth [PO] BID [3×1 g capsules and 2×1 gcapsules])

Day 1, every 2 weeks:

Bevacizumab (5 mg/kg IV)

Day 1, every 4 weeks (every other treatment cycle):

Fulvestrant (500 mg IM)

Days 1-5 and 8-12, every 2 weeks:

Cyclophosphamide (50 mg PO twice a day [BID]).

Days 1, 3, 5, 8, 10 and 12, every 2 weeks:

5-FU (400 mg/m2 continuous IV infusion over 24 hours)

Leucovorin (20 mg/m2 IV bolus)

Day 1 and 8, every 2 weeks:

Nab-paclitaxel (100 mg IV)

Cisplatin (40 mg/m2 IV)

Day 5, 19, 33 (every 2 weeks for 3 doses then every 8 weeks thereafter):

ETBX-011, ETBX-021, ETBX-051, ETBX-061 (5×10¹¹ virus particles[VI:]/vaccine/dose subcutaneously [SC])

GI-4000, GI-6207, GI-6301, (40 yeast units [YU]/vaccine/dose SC), 2hours after administration of the Ad5-based vaccines

Prospective tumor molecular profiling will determine whether ETBX-021and GI-4000 will be administered, as described above.

Day 8, every week:

Cetuximab (250 mg IV)

Day 8, every 2 weeks:

Nivolumab (3 mg/kg IV over 1 hour) or avelumab (10 mg/kg IV over 1hour).

Day 8, 22, 36, 50 (every 2 weeks for 4 doses):

SBRT (not to exceed 8 Gy, exact dose to be determined by the radiationoncologist)

Day 9, every 2 weeks:

ALT-803 (10 μg/kg SC 30 minutes prior to aNK infusion)

Day 9 and 11, every 2 weeks:

haNK (2×10⁹ cells/dose IV)

Maintenance Phase:

The duration of the maintenance phase will be up to 1 year followingcompletion of the last treatment in the induction phase. The maintenancephase will comprise repeated 2-week cycles. The treatment regimen ofomega-3-acid ethyl esters, cyclophosphamide, capecitabine,nab-paclitaxel, bevacizumab, ALT-803, haNK cells, Ad5-based vaccines(ETBX-011, ETBX-021, ETBX-051, and ETBX-061), yeast-based vaccines(GI-4000, GI-6207, and GI-6301), nivolumab or avelumab, fulvestrant, andcetuximab will be repeated every 2 weeks.

The maintenance phase of the treatment will be conducted in accordancewith the following dosing regimen:

Daily:

Omega-3-acid ethyl esters (by mouth [PO] BID [3×1 g capsules and 2×1 gcapsules])

Day 1, every 2 weeks:

Bevacizumab (5 mg/kg IV)

Nab-paclitaxel (100 mg IV)

Nivolumab (3 mg/kg IV over 1 hour) or avelumab (10 mg/kg IV over 1hour).

Cetuximab (250 mg IV)

Day 1, every 4 weeks (every other treatment cycle):

Fulvestrant (500 mg IM)

Days 1-5 and 8-12, every 2 weeks:

Capecitabine (650 mg/m2 PO BID)

Cyclophosphamide (50 mg PO BID)

Day 2, every 2 weeks:

ALT-803 (10 μg/kg SC) (30 minutes prior to aNK infusion)

haNK (2×10⁹ cells/dose IV)

Day 5, every 8 weeks thereafter:

ETBX-011, ETBX-021, ETBX-051, ETBX-061 (5×10¹¹ VP/vaccine/dose SC)

GI-4000, GI-6207, GI-6301 (40 YU/vaccine/dose SC), 2 hours afteradministration of Ad-5 based vaccines.

Prospective tumor molecular profiling will determine whether ETBX-021and GI-4000 will be administered, as described above. FIG. 8schematically illustrates the exemplary treatment protocol.

Tumor Molecular Profiling: Genomic sequencing of tumor cells from tissuerelative to non-tumor cells from whole blood will be conducted toidentify tumor-specific genomic variances that may contribute to diseaseprogression and/or response to treatment. RNA sequencing will beconducted to provide expression data and give relevance to DNAmutations. Quantitative proteomics analysis will be conducted todetermine the absolute amounts of specific proteins, to confirmexpression of genes that are correlative of disease progression and/orresponse, and to determine cutoff values for response. All genomic,transcriptomic, and proteomic molecular analyses will be exploratory,except for the prospective tumor molecular analysis of HER2 expressionby quantitative proteomics and analysis of Ras mutational status bygenomic sequencing to determine whether ETBX-021 and GI-4000 will beadministered.

Follow-up Analyses/Sample Collection and Analysis: Tumor molecularprofiling will be performed on FFPE tumor tissue and whole blood(subject-matched normal comparator against the tumor tissue) bynext-generation sequencing and mass spectrometry-based quantitativeproteomics. Tumor tissue and whole blood samples will be collected andshipped in accordance with the instruction cards included in the TissueSpecimen Kit and Blood Specimen Kit. The specimen requirements andprocedural instructions for sample collection are described in theNantOmics Sample Collection Manual. An FFPE tumor tissue specimen isrequired for the extraction of tumor DNA, tumor RNA, and tumor protein.A whole blood sample is required for the extraction of subject normalDNA. Tumor tissue and whole blood will be processed in CLIA-certifiedand CAP-accredited clinical laboratories.

Exploratory Immunology Analysis: One aim of immunotherapy treatment isto generate antigen-specific antitumor immune responses. Exploratoryimmunology analysis will be used to provide a preliminary assessment ofimmune responses induced by the treatments. Blood samples for immuneanalysis will be collected from subjects at screening and every month inthe induction phase and every 2 months in the maintenance phase duringroutine blood draws. PBMCs isolated by Ficoll-Hypaque density gradientseparation will be analyzed for antigen-specific immune responses usingELISpot assays for IFN-γ or granzyme B secretion after exposure to thefollowing tumor-associated antigen peptides: CEA, Brachyury, and MUC1,and if ETBX-021 and GI-4000 are administered, HER2 and mutant Ras,respectively. Flow cytometry will be utilized to assess T-cell responsesusing intracellular cytokine staining assay for IFN-γ or TNF-αexpression after exposure to the tumor-associated antigen peptides. Flowcytometry analysis for the expression of CD107a on cells will beutilized to test for degranulating cells such as CD8+ T cells and NKcells. PBMCs will be stimulated in vitro with overlapping 15-mer peptidepools encoding the tumor-associated antigens mentioned above. Controlpeptide pools will involve the use of irrelevant antigen peptide poolsas a negative control and CEFT peptide mix as a positive control. CEFTis a mixture of peptides of CMV, Epstein-Barr virus, influenza, andtetanus toxin. Post-stimulation analyses of CD4+ and CD8+ T cells willinvolve the production of IFN-γ, TNF-α, and CD107a expression. Sera willbe analyzed for antibodies directed to the aforementionedtumor-associated antigens, neutralizing antibody titer to adenovirus(serotype 5), and for potential antibody development against theIL-15N72D:IL-15RαSu/IgG1 Fc complex.

Circulating Tumor DNA and RNA Assays: Tumors evolve during therapy, anddrug-resistant cells emerge, which are difficult to detect and may causethe tumor to become resistant to the initial treatment. Blood-basedtesting for ctDNA and ctRNA can track the emergence of drug-resistanttumor cells and can identify new drug targets and treatment options forpatients. Whole blood will be collected at screening and every month inthe induction phase and every 2 months in the maintenance phase duringroutine blood draws for the analysis of ctDNA and ctRNA. Expressionlevels of specific tumor- and immune-related analytes in ctDNA and ctRNAwill be measured by qPCR and analyzed for correlations with subjectoutcomes.

Merkel Cell Carcinoma:

Skin cancer is the most common malignancy diagnosed in the UnitedStates, with more than 2 million Americans diagnosed annually. Merkelcell carcinoma (MCC) is a rare and aggressive type of skin cancer thatwas thought to arise from Merkel cells located between the dermal andepidermal layers of the skin. Approximately 1,500 new cases wereexpected in 2007 in the US. MCC is more common in whites,individuals >65 years old, men, and subjects with acquired (e.g., HIVinfection) or iatrogenic immune suppression (e.g., due to treatment ofautoimmune diseases). Ultraviolet exposure is an independent risk factorfor the disease and may contribute to the rising incidence of MCC.

MCC that is confined to the skin has a good prognosis and can often becured by surgery alone. The 5 year OS rate for subjects presenting withlocal disease is 66% for tumors <2 cm and 51% for tumors >2 cm.Metastatic MCC has a much poorer prognosis, with 5-year OS of 39% forsubjects with regional lymph node involvement and 18% for those withmetastases to distant organs. Advanced disease stage, location in theperineum or lower extremities, male gender, advanced age (>60 yearsold), immunosuppression, comorbid factors, high mitotic rate, andangiolymphatic invasion are associated with poor prognosis. Surgicalresection is the cornerstone of therapy for MCC, with the goal ofestablishing clear surgical margins by wide local excision. Adjuvantradiation therapy to the primary tumor bed in subjects with stage I/IIMCC has been shown to improve OS, however, neither systemic chemotherapynor radiation therapy in subjects with stage III disease improves OS,although some studies suggest chemotherapy may increase survival insubjects with advanced MCC.

Cytotoxic chemotherapy is often used to treat metastatic MCC. A minorityof subjects treated with chemotherapy respond well to treatment, butresponses are usually transient and rarely lead to significant increasesin survival time. Adjuvant treatment with etoposide and carboplatin hasnot been associated with OS benefit for subjects with advancedloco-regional disease. Some studies have demonstrated high objectiveantitumor responses (>50%) using cytotoxic chemotherapy(etoposide-carboplatin andcyclophosphamide-doxorubicin-vincristine-prednisone have been the mostfrequently used) in subjects with metastatic MCC. However, theseresponses are rarely durable, and are associated with a median OS of 9months. Moreover, high rates of chemotoxic death were associated withfirst-line treatments. At present, limited data exist to guide treatmentdecisions regarding chemotherapy and radiotherapy, and often decisionsare made based on comorbidities and consideration of AEs. For subjectswith metastatic MCC, limited treatment options and limited efficacy ofavailable therapies emphasize the need for additional therapeuticoptions.

In general, the overall goals of the Merkel Cell Carcinoma vaccinetreatment are to maximize ICD and augment and maintain the innate andadaptive immune responses against cancer cells. The rationale for theselection of agents is summarized in Table 2 in which 5-FU is5-fluorouracil; haNK is high-affinity activated natural killer; ICD isimmunogenic cell death; SBRT is stereotactic body radiation therapy, andTME is tumor microenvironment.

Enhancing Mitigating Inducing and Conditioning Innate MaintainingImmunosuppression Coordinating Dendritic Immune Immune Agent in the TMEICD Signals and T Cells Responses Responses ALT-803 X X Avelumab XBevacizumab X X Capecitabine X X Cisplatin X Cyclophosphamide X XETBX-051 X ETBX-061 X 5-FU X X GI-6301 X haNK cells X Nab-paclitaxel X XOmega-3-acid ethyl esters X SBRT X X

FIG. 9 exemplarily and schematically depicts the mechanism(s) by whicheach agent impacts the immune system, consequently leading to ICD. Bycombining agents that simultaneously (or sequentially) target distinctbut complementary mechanisms that enable tumor growth, the treatmentregimen aims to maximize anticancer activity and prolong the duration ofresponse to treatment.

To that end, contemplated MCC treatments combine LDMC, bevacizumab, acancer vaccine, low-dose radiation therapy, an IL-15 superagonist, NKcell therapy, and a checkpoint inhibitor. Such treatment is thought tomaximize ICD and augment and maintain the innate and adaptive immuneresponses against cancer cells. More specifically, the treatment regimenis set up to interrupt the escape phase of immunoediting by: (a)Mitigating immunosuppression in the TME. LDMC will be used to reduce thedensity of Tregs, MDSCs, and M2 macrophages contributing toimmunosuppression in the TME. Bevacizumab will be used to causemorphological changes in the TME to promote lymphocyte trafficking; (b)Inducing and coordinating ICD signals. LDMC and low-dose radiationtherapy will be used to increase the antigenicity of tumor cells.Bevacizumab will be used to alter the TME, which allows for moreefficient antigen-specific T-cell responses and makes tumor cells moresusceptible to ICD. Omega-3-acid ethyl esters enhances ICD withoutincreasing toxicity; (c) Conditioning dendritic and T cells. A cancervaccine and an IL-15 superagonist will be used to enhance tumor-specificcytotoxic T-cell responses; (d) Enhancing innate immune responses. NKcell therapy will be used to augment the innate immune system. An IL-15superagonist will be used to enhance the activity of endogenous andintroduced NK cells. Hypofractionated-dose radiation therapy will beused to upregulate tumor cell NK ligands to enhance tumor cytotoxicityof NK cells; and (e) Maintaining immune responses. A checkpointinhibitor will be used to promote long-term anticancer immune responses.

The MCC vaccine treatment will be conducted in 2 phases: an inductionphase and a maintenance phase. The purpose of the induction phase is tostimulate immune responses against tumor cells and mitigateimmunosuppression in the TME. The purpose of the maintenance phase is tosustain ongoing immune system activity against tumor cells, creatingdurable treatment responses. Exemplary use and timing of ofadministration of contemplated compounds and compositions for theinduction phase and the maintenance phase are shown in FIG. 10 and FIG.11, respectively. Therefore, the following agents and compositions arepreferably used for the induction and maintenance phases:

1. ALT-803, recombinant human super agonist interleukin-15 (IL-15)complex (also known as IL 15N72D:IL-15RαSu/IgG1 Fc complex); 2. Avelumab(BAVENCIO® injection, for IV use); 3. Bevacizumab (AVASTIN® solution forIV infusion); 4. Capecitabine (XELODA® tablets, for oral use); 5.Cisplatin (CISplatin injection); 6.Cyclophosphamide (CYCLOPHOSPHAMIDECapsules, for oral use); 7. ETBX-051 (Ad5 [E1-, E2b-]-Brachyury); 8.ETBX-061 (Ad5 [E1-, E2b-]-MUC1); 9. 5-FU (Fluorouracil Injection, for IVuse only); 10. GI-6301 (Brachyury yeast vaccine); 11. haNK™, NK-92[CD16.158V, ER IL-2], Suspension for Intravenous Infusion (haNK™ forInfusion); 12. Leucovorin (LEUCOVORIN Calcium for Injection, for IV orIM use); 13. Nab-paclitaxel (ABRAXANE® for Injectable Suspension[paclitaxel protein-bound particles for injectable suspension][albumin-bound]); 14. Omega-3-acid ethyl esters (Lovaza capsules, fororal use); and 15. SBRT.

More specifically, an exemplary treatment protocol for MCC willtypically include the following steps, phases, compounds andcompositions:

Tumors will be assessed at screening, and tumor response will beassessed every 8 weeks during the induction phase and every 12 weeksduring the maintenance phase by computed tomography (CT), magneticresonance imaging (MRD, or positron emission tomography-computedtomography (PET CT) of target and non-target lesions in accordance withResponse Evaluation Criteria in Solid Tumors (RECIST) Version 1.1 andimmune-related response criteria (irRC).

Tumor biopsies and exploratory tumor molecular profiling will beconducted at screening, at the end of the initial induction phase (8weeks after the start of treatment), and during potential prolongedinduction and maintenance phases (depending on response). Separate bloodtubes will be collected every month in the induction phase and every 2months in the maintenance phase during routine blood draws forexploratory immunology and ctDNA/ctRNA analyses.

Induction Phase: The induction phase will comprise of repeated 2 weekcycles. The treatment regimen of omega-3-acid ethyl esters,cyclophosphamide, cisplatin, 5 FU/leucovorin, nab-paclitaxel,bevacizumab, ALT-803, haNK cells, Ad5-based vaccines (ETBX-051 andETBX-061), GI-6301 yeast vaccine and avelumab will be repeated every 2weeks. Concurrent SBRT will be given during the first four 2-weekcycles. Radiation will be administered to all feasible tumor sites usingSBRT. Contemplated techniques include linear-accelerator based therapies(3D and intensity-modulated radiation therapy [IMRT]). Specifically, theinduction phase of the treatment will be conducted in accordance withthe following dosing regimen:

Day 1, daily:

Omega-3-acid ethyl esters (5×1 g capsules by mouth [PO])

Day 1, every 2 weeks:

Bevacizumab (5 mg/kg IV)

Days 1-5 and 8-12, every 2 weeks:

Cyclophosphamide (50 mg PO twice a day [BID]).

Days 1, 3, 5, 8, 10 and 12, every 2 weeks:

5-FU (400 mg/m2 as a continuous IV infusion over 24 hours)

Leucovorin (20 mg/m2 IV bolus)

Day 1 and 8, every 2 weeks:

Nab-paclitaxel (100 mg IV)

Cisplatin (40 mg/m2 IV)

Day 5, 19, 33 (every 2 weeks for 3 doses then every 8 weeks thereafter):

ETBX-051, ETBX-061 (5×10¹¹ virus particles [VI:]/vaccine/dosesubcutaneously [SC])

GI-6301 (40 yeast units [YU]/dose SC), 2 hours after administration ofAd5-based vaccines

Day 8, every 2 weeks:

Avelumab (10 mg/kg IV over 1 h)

Day 8, 22, 36, 50 (every 2 weeks for 4 doses):

SBRT (not to exceed 8 Gy, exact dose to be determined by the radiationoncologist)

Day 9, every 2 weeks:

ALT-803 (10 μg/kg SC 30 minutes prior to haNK infusion)

Day 9 and 11, every 2 weeks:

haNK (2×10⁹ cells/dose IV)

Maintenance Phase: The maintenance phase of the treatment will beconducted in accordance with the following dosing regimen:

Day 1, daily:

Omega-3-acid ethyl esters (5×1 g capsules PO)

Day 1, every 2 weeks:

Bevacizumab (5 mg/kg IV)

Nab-paclitaxel (100 mg IV)

Avelumab (10 mg/kg IV over 1 hour)

Days 1-5 and 8-12, every 2 weeks:

Cyclophosphamide (50 mg PO BID)

Capecitabine (650 mg/m2 PO BID)

Day 2, every 2 weeks:

ALT-803 (10 μg/kg SC) (30 minutes prior to haNK infusion)

haNK (2×10⁹ cells/dose IV)

Day 5, every 8 weeks thereafter:

ETBX-051, ETBX-061 (5×10¹¹ VP/vaccine/dose SC)

GI-6301 (40 YU/dose SC), 2 hours after administration of Ad5-basedvaccines

FIG. 12 schematically illustrates the exemplary treatment protocol.

Tumor molecular profiling before, during, and after treatment will beperformed on FFPE tumor tissue and whole blood (subject-matched normalcomparator against tumor tissue) by next-generation sequencing and massspectrometry-based quantitative proteomics.

Follow-up analyses/Sample collection and Analysis: Most typically, anFFPE tumor tissue specimen is required for the extraction of tumor DNA,tumor RNA, and tumor protein, and a whole blood sample is required forthe extraction of subject normal DNA. Tumor tissue and whole blood willbe processed in CLIA-certified and CAP-accredited clinical laboratories.

Exploratory Immunology Analysis: One aim of immunotherapy treatment isto generate antigen-specific antitumor immune responses. Exploratoryimmunology analysis will be used to provide a preliminary assessment ofimmune responses induced by the treatments. Blood samples for immuneanalysis will be collected from subjects at screening/baseline and everymonth in the induction phase and every 2 months in the maintenance phaseduring routine blood draws. A sample of 10.0 mL is required at the blooddraw. PBMCs isolated by Ficoll-Hypaque density gradient separation willbe analyzed for antigen-specific immune responses using ELISpot assaysfor IFN-γ or granzyme B secretion after exposure to Brachyury and MUC1peptides. Flow cytometry will be utilized to assess T cell responsesusing intracellular cytokine staining assay for IFN-γ or TNF-αexpression after exposure to Brachyury and MUC1 peptides. Flow cytometryanalysis for the expression of CD107a on cells will be utilized to testfor degranulating cells such as CD8+ T cells and NK cells (Kalman 1996).PBMCs will be stimulated in vitro with overlapping 15-mer peptide poolsencoding Brachyury and MUC1. Control peptide pools will involve the useof irrelevant antigen peptide pools as a negative control and CEFTpeptide mix as a positive control. CEFT is a mixture of peptides ofcytomegalovirus, EBV, influenza, and tetanus toxin. Post-stimulationanalyses of CD4 and CD8 T cells will involve the production of IFN-γ,TNF-α, and CD107a expression. Sera will be analyzed for Brachyury- andMUC1 directed antibodies, neutralizing antibody titer to adenovirus(serotype 5), and for potential antibody development against theIL-15N72D:IL-15RαSu/IgG1 Fc complex.

Circulating Tumor DNA and RNA Assays: Tumors evolve during therapy, anddrug-resistant cells emerge, which are difficult to detect and may causethe tumor to become resistant to the initial treatment. Blood-basedtesting for ctDNA and ctRNA can track the emergence of drug-resistanttumor cells and can identify new drug targets and treatment options forpatients. Whole blood will be collected at screening/baseline and everymonth in the induction phase and every 2 months in the maintenance phaseduring routine blood draws for the analysis of ctDNA and ctRNA; a sampleof 20.0 mL is required at the blood draw. Whole blood will be drawn intoCell-Free DNA BCT® tubes or Cell-Free RNA BCT® tubes containing DNA orRNA stabilizers, respectively. Expression levels of specific tumor- andimmune-related analytes in ctDNA and ctRNA will be measured by qPCR andanalyzed for correlations with subject outcomes.

Melanoma:

Skin cancer is the most common malignancy diagnosed in the US, with morethan 2 million Americans diagnosed annually. Three main types of skincancer exist: basal cell carcinoma, squamous cell carcinoma (SCC),collectively referred to as non-melanoma skin cancer, and melanoma.Melanoma is a malignant tumor of melanocytes and accounts for only about1% of skin cancers, but the vast majority of skin cancer deaths. Anestimated 87,110 new melanoma cases will be diagnosed in the US in 2017with an estimated 9,730 deaths.

Melanoma incidence is rising rapidly in the US, and incidence rates havedoubled from 1982 to 2011. More than 90% of melanoma cases have beenattributed to excessive UV exposure, and increasing incidence rates arethought to reflect rising cumulative UV exposure. In addition to sunexposure, risk factors for developing melanoma include skinpigmentation, with lighter skin conferring higher risk. Melanoma is 20times more common in whites than in African-Americans. A positive familyhistory of melanoma, and the presence of some rare genetic mutations arealso associated with higher risk for the disease.

Treatment for early-stage melanoma is largely effective, and forpatients with localized disease, 5-year survival rates exceed 90%.Treatment options for early-stage melanoma focus on excision of thetumor while achieving positive tumor margins. However, for patients withmetastatic or recurrent disease, prognoses are much poorer, and 5-yearsurvival rates have historically been less than 10%, and median OS lessthan 1 year.

Treatment options for unresectable late-stage, and recurrent melanomainclude intralesional therapy, immunotherapy, signal transductioninhibitors, chemotherapy, and palliative local therapy. Newimmunotherapies have offered novel treatment options for patients withadvanced stage melanoma, and treatment with these agents has resulted indurable responses in a subset of patients. Immunotherapies currentlyapproved for treatment of advanced melanoma include interleukin-2 (IL-2)and the checkpoint inhibitors ipilimumab, nivolumab, and pembrolizumab.A retrospective analysis of 8 studies of subjects with metastaticmelanoma treated with high-dose IL-2 showed an overall ORR of 16%. Ofthe subjects who responded, 28% remained progression free at a medianfollow-up of 62 months. However, the high toxicities associated withIL-2, including capillary leak syndrome, limit its widespread use. Inrandomized trials, two approaches in particular, checkpoint inhibitionand inhibition of the mitogen-activated protein kinase (MAPK) signaltransduction pathway, have demonstrated improvement in OS when comparedto dacarbazine monotherapy, which has long been the SoC for advancedmelanoma. In clinical trials, treatment with dacarbazine has resulted inORRs of 10-20%, but has not been associated with improvements in OS.

Signal transduction inhibitors that target the MAPK pathway,specifically, V-raf murine sarcoma viral oncogene homolog B1 (BRAF) andmitogen-activated ERK- (extracellular signal-regulated kinase)activating kinase (MEK) have also been investigated as treatment inpatients with unresectable or advanced disease. BRAF gene mutations arethe most frequent mutations in cutaneous melanoma. Approximately 40% to60% of malignant melanomas harbor a single nucleotide mutation in BRAF;the most commonly found is a valine to glutamic acid substitution atposition 600 (BRAF V600E). Vemurafenib, a selective BRAF V600E kinaseinhibitor, has demonstrated improvement in both PFS and OS in patientswith advanced disease, although its indication is limited to patientsthat have the BRAF V600E mutation as detected by an FDA-approved test.Dabrafenib is another selective inhibitor of BRAF that has resulted inimprovement in PFS when compared to dacarbazine. The MEK inhibitors,trametinib and cobimetinib, have also been approved for treatment ofpatients with unresectable or metastatic melanoma. Monotherapy treatmentwith trametinib showed an improvement in PFS compared to thechemotherapy group (either dacarbazine or paclitaxel). Similarly,cobimetinib in combination with vemurafenib showed a significantincrease in PFS over vermurafenib treatment alone.

Although treatment options for unresectable late-stage and recurrentmelanoma have increased, neither checkpoint inhibition nor MAPK pathwayinhibition appear to be curative when used as monotherapy.

In general, the overall goals of the melanoma Vaccine treatment are tomaximize ICD and augment and maintain the innate and adaptive immuneresponses against cancer cells. The rationale for the selection ofagents included in contemplated treatments is summarized in Table 3 inwhich a) denotes either avelumab or nivolumab will be administered; b)denotes Capecitabine is metabolized to 5-FU; and c) denotes thatLeucovorin potentiates the activity of 5-FU.

Enhancing Mitigating Inducing and Conditioning Innate MaintainingImmunosuppression Coordinating Dendritic Immune Immune Agent in the TMEICD Signals and T Cells Responses Responses ALT-803 X XAvelumab/nivolumab^(i)) X Bevacizumab X X Capecitabine^(ii)) X XCisplatin X Cyclophosphamide X X ETBX-011 X ETBX-051 X ETBX-061 X5-FU/leucovorin^(iii)) X X GI-6207 X GI-6301 X haNK cells XNab-paclitaxel X X Omega-3-acid ethyl esters X SBRT X X

FIG. 13 schematically and exemplarily depicts the mechanism(s) by whicheach agent impacts the immune system, consequently leading to ICD. Bycombining agents that simultaneously target distinct but complementarymechanisms that enable tumor growth, the treatment regimen aims tomaximize anticancer activity and prolong the duration of response totreatment.

To that end, contemplated melanoma treatments combine LDMC, bevacizumab,a cancer vaccine, low-dose radiation therapy, an IL-15 superagonist, NKcell therapy, and a checkpoint inhibitor. The overall goals of thetreatment regimen are to maximize ICD and augment and maintain theinnate and adaptive immune responses against cancer cells. Morespecifically, the treatment is designed to interrupt the escape phase ofimmunoediting by: (a) Mitigating immunosuppression in the TME. LDMC willbe used to reduce the density of Tregs, MDSCs, and M2 macrophagescontributing to immunosuppression in the TME. Bevacizumab will be usedto cause morphological changes in the TME to promote lymphocytetrafficking; (b) Inducing and coordinating ICD signals. LDMC andlow-dose radiation therapy will be used to increase the antigenicity oftumor cells. Bevacizumab will be used to alter the TME, which allows formore efficient antigen-specific T-cell responses and makes tumor cellsmore susceptible to ICD. Omega-3-acid ethyl esters enhances ICD withoutincreasing toxicity; (c) Conditioning dendritic and T cells. A cancervaccine and an IL-15 superagonist will be used to enhance tumor-specificcytotoxic T-cell responses; (d) Enhancing innate immune responses. NKcell therapy will be used to augment the innate immune system. An IL-15superagonist will be used to enhance the activity of endogenous andintroduced NK cells. Hypofractionated-dose radiation therapy will beused to upregulate tumor cell NK ligands to enhance tumor cytotoxicityof NK cells; and (e) Maintaining immune responses. A checkpointinhibitor will be used to promote long-term anticancer immune responses.

The melanoma vaccine treatment will be conducted in 2 phases: aninduction phase and a maintenance phase. The purpose of the inductionphase is to stimulate immune responses against tumor cells and mitigateimmunosuppression in the TME. The purpose of the maintenance phase is tosustain ongoing immune system activity against tumor cells, creatingdurable treatment responses. Exemplary use and timing of administrationof contemplated compounds and compositions for the induction phase andthe maintenance phase are shown in FIG. 14 and FIG. 15, respectively.Therefore, the following agents and compositions are preferably used forthe induction and maintenance phases:

1. ALT-803, recombinant human super agonist interleukin-15 (IL-15)complex (also known as IL 15N72D:IL-15RαSu/IgG1 Fc complex); 2. Avelumab(BAVENCIO® injection, for IV use); 3. Bevacizumab (AVASTIN® solution forIV infusion); 4.Capecitabine (XELODA® tablets, for oral use); 5.Cisplatin (Cis-platin injection); 6. Cyclophosphamide (CYCLOPHOSPHAMIDECapsules, for oral use); 7. ETBX-011 (Ad5 [E1-, E2b-]-CEA); 8. ETBX-051(Ad5 [E1-, E2b-]-Brachyury); 9. ETBX-061 (Ad5 [E1-, E2b-]-MUC1); 10.5-FU (Fluorouracil Injection, for IV use only); 11. GI-6207 (CEA yeastvaccine); 12. GI-6301 (Brachyury yeast vaccine); 13. haNK™, NK-92[CD16.158V, ER IL-2], Suspension for Intravenous Infusion (haNK™ forInfusion); 14. Leucovorin (LEUCOVORIN Calcium for Injection, for IV orIM use); 15. Nab-paclitaxel (ABRAXANE® for Injectable Suspension[paclitaxel protein-bound particles for injectable suspension][albumin-bound]); 16. Nivolumab (OPDIVO® injection, for IV use); 17.Omega-3-acid ethyl esters (Lovaza capsules, for oral use); 18. SBRT.

More specifically, an exemplary treatment protocol for melanoma willtypically include the following steps, phases, compounds, andcompositions:

Tumor biopsies and exploratory tumor molecular profiling will beconducted at screening, at the end of the initial induction phase (8weeks after the start of treatment), and during potential prolongedinduction and maintenance phases (depending on response). Separate bloodtubes will be collected every month in the induction phase and every 2months in the maintenance phase during routine blood draws forexploratory immunology and ctDNA/ctRNA analyses.

Tumors will be assessed at screening, and tumor response will beassessed every 8 weeks during the induction phase and every 12 weeksduring the maintenance phase by computed tomography (CT), magneticresonance imaging (MRI), or positron emission tomography-computedtomography (PET CT) of target and non-target lesions in accordance withResponse Evaluation Criteria in Solid Tumors (RECIST) Version 1.1 andimmune-related response criteria (irRC).

Induction Phase: The induction phase will comprise repeated 2 weekcycles. The treatment regimen of ALT-803, Ad5-based vaccines (ETBX-011,ETBX-051, and ETBX-061), yeast-based vaccines (GI-6207 and GI-6301),haNK cells, avelumab or nivolumab, bevacizumab, cisplatin,cyclophosphamide, 5 FU/leucovorin, nab-paclitaxel, and omega-3-acidethyl esters will be repeated every 2 weeks. Concurrent SBRT will begiven during the first four 2-week cycles. Radiation will beadministered to all feasible tumor sites using SBRT Specifically, anexemplary induction phase of melanoma treatment will be conducted inaccordance with the following dosing regimen:

Daily:

Omega-3-acid ethyl esters (by mouth [PO] twice a day [BID] [3×1 gcapsules and 2×1 g capsules])

Day 1, every 2 weeks:

Bevacizumab (5 mg/kg IV)

Days 1-5 and 8-12, every 2 weeks:

Cyclophosphamide (50 mg PO BID).

Days 1, 3, 5, 8, 10 and 12, every 2 weeks:

5-FU (400 mg/m2 as a continuous IV infusion over 24 hours)

Leucovorin (20 mg/m2 IV bolus)

Day 1 and 8, every 2 weeks:

Nab-paclitaxel (100 mg IV)

Cisplatin (40 mg/m2 IV)

Day 5, 19, 33 (every 2 weeks for 3 doses then every 8 weeks thereafter):

ETBX-011, ETBX-051, ETBX-061 (5×10¹¹ virus particles [VI:]/vaccine/dosesubcutaneously [SC])

GI-6207, GI-6301 (40 yeast units [YU]/vaccine/dose SC), 2 hours afteradministration of Ad5-based vaccines

Day 8, every 2 weeks:

Avelumab (10 mg/kg IV over 1 h) or nivolumab (3 mg/kg IV over 1 h).

Day 8, 22, 36, 50 (every 2 weeks for 4 doses):

SBRT (not to exceed 8 Gy, exact dose to be determined by the radiationoncologist)

Day 9, every 2 weeks:

ALT-803 (10 μg/kg SC 30 minutes prior to haNK infusion)

Day 9 and 11, every 2 weeks:

haNK (2×10⁹ cells/dose IV)

Maintenance Phase: The duration of the maintenance phase will be up toone year following completion of the last treatment in the inductionphase. The maintenance phase will comprise repeated 2-week cycles. Thetreatment regimen of ALT-803, Ad5 based vaccines (ETBX-011, ETBX 051,and ETBX 061), yeast-based vaccines (GI-6207 and GI-6301), haNK cells,avelumab or nivolumab, bevacizumab, capecitabine, cyclophosphamide,nab-paclitaxel, and omega-3-acid ethyl esters will be repeated every 2weeks.

The maintenance phase of the treatment will be conducted in accordancewith the following dosing regimen:

Daily:

Omega-3-acid ethyl esters (PO BID [3×1 g capsules and 2×1 g capsules])

Day 1, every 2 weeks:

Bevacizumab (5 mg/kg IV)

Nab-paclitaxel (100 mg IV)

Avelumab (10 mg/kg IV over 1 h) or nivolumab (3 mg/kg IV over 1 hour).

Days 1-5 and 8-12, every 2 weeks:

Cyclophosphamide (50 mg PO BID)

Capecitabine (650 mg/m2 PO BID)

Day 2, every 2 weeks:

ALT-803 (10 μg/kg SC 30 minutes prior to haNK infusion)

haNK (2×10⁹ cells/dose IV)

Day 5, every 8 weeks thereafter:

ETBX-011, ETBX-051, ETBX-061 (5×10¹¹ VP/vaccine/dose SC)

GI-6301 (40 YU/dose SC), 2 hours after administration of Ad5-basedvaccines.

FIG. 16 schematically illustrates the exemplary treatment protocol.

Tumor Molecular Profiling: Genomic sequencing of tumor cells from tissuerelative to non-tumor cells from whole blood will be conducted toidentify tumor-specific genomic variances that may contribute to diseaseprogression and/or response to treatment. RNA sequencing will beconducted to provide expression data and give relevance to DNAmutations. Quantitative proteomics analysis will be conducted todetermine the absolute amounts of specific proteins, to confirmexpression of genes that are correlative of disease progression and/orresponse, and to determine cutoff values for response.

Follow-up Analyses/Sample Collection and Analysis: Tumor molecularprofiling will be performed on FFPE tumor tissue and whole blood(subject-matched normal comparator against the tumor tissue) bynext-generation sequencing and mass spectrometry-based quantitativeproteomics. Collection of tumor tissue and whole blood at screening andat the end of the initial induction phase (8 weeks after the start oftreatment) is contemplated.

Tumor tissue and whole blood samples will be collected and shipped inaccordance with the instruction cards included in the Tissue SpecimenKit and Blood Specimen Kit. An FFPE tumor tissue specimen is requiredfor the extraction of tumor DNA, tumor RNA, and tumor protein. A wholeblood sample is required for the extraction of subject normal DNA. Tumortissue and whole blood will be processed in CLIA-certified andCAP-accredited clinical laboratories.

Exploratory Immunology Analysis: One aim of immunotherapy treatment isto generate antigen-specific antitumor immune responses. Exploratoryimmunology analysis will be used to provide a preliminary assessment ofimmune responses induced by the treatments. Blood samples for immuneanalysis will be collected from subjects at screening and every month inthe induction phase and every 2 months in the maintenance phase duringroutine blood draws. A sample of 10.0 mL is required at the blood draw.PBMCs isolated by Ficoll-Hypaque density gradient separation will beanalyzed for antigen-specific immune responses using ELISpot assays forIFN-γ or granzyme B secretion after exposure to CEA, Brachyury, and MUC1peptides. Flow cytometry will be utilized to assess T cell responsesusing intracellular cytokine staining assay for IFN-γ or TNF-αexpression after exposure to CEA, Brachyury, and MUC1 peptides. Flowcytometry analysis for the expression of CD107a on cells will beutilized to test for degranulating cells such as CD8+ T cells and NKcells (Kannan 1996). PBMCs will be stimulated in vitro with overlapping15-mer peptide pools encoding CEA, Brachyury, and MUC1. Control peptidepools will involve the use of irrelevant antigen peptide pools as anegative control and CEFT peptide mix as a positive control. CEFT is amixture of peptides of cytomegalovirus, EBV, influenza, and tetanustoxin. Post-stimulation analyses of CD4 and CD8 T cells will involve theproduction of IFN-γ, TNF-α, and CD107a expression. Sera will be analyzedfor CEA-, Brachyury-, and MUC1 directed antibodies, neutralizingantibody titer to adenovirus (serotype 5), and for potential antibodydevelopment against the IL-15N72D:IL-15RαSu/IgG1 Fc complex.

Circulating Tumor DNA and RNA Assays: Tumors evolve during therapy, anddrug-resistant cells emerge, which are difficult to detect and may causethe tumor to become resistant to the initial treatment. Blood-basedtesting for ctDNA and ctRNA can track the emergence of drug-resistanttumor cells and can identify new drug targets and treatment options forpatients. Whole blood will be collected at screening and every month inthe induction phase and every 2 months in the maintenance phase duringroutine blood draws for the analysis of ctDNA and ctRNA. Expressionlevels of specific tumor- and immune-related analytes in ctDNA and ctRNAwill be measured by qPCR and analyzed for correlations with subjectoutcomes.

Non-Hodgkin Lymphoma:

NHL is a highly prevalent disease in the US with a projected 72,240 newcases diagnosed in 2017, which accounts for approximately 4% of allcancers. This disease is the ninth-leading cause of cancer-relateddeaths with an estimate of 20,140 deaths in 2017. NHL can be classifiedas B-cell lymphomas or T-cell lymphomas. About 85% of NHL cases in theUS are B-cell lymphomas. B-cell lymphomas comprise various subtypes,including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma,small lymphocytic lymphoma, mantle cell lymphoma, marginal zonelymphomas, Burkitt lymphoma, and lymphoplasmacytic lymphoma. Of theB-cell lymphomas, DLBCL is the most common and is typically anaggressive disease. Follicular lymphoma, small lymphocytic lymphoma,marginal zone lymphoma, and lymphoplasmacytic lymphoma tend to beindolent diseases. Less than 15% of NHL cases in the US are T-celllymphomas. Similar to B-cell lymphomas, there are many subtypes ofT-cell lymphomas, which include precursor T-lymphoblastic lymphoma andperipheral T-cell lymphomas. Patients with NHL typically present withadvanced stage (III/IV) disease, and many are initially asymptomatic.

Treatment of NHL varies based on the type and extent of disease andincludes chemotherapy, immunotherapy, targeted therapy, radiationtherapy, and stem cell transplant. Standard first-line therapy ofCD20-positive NHL involves treatment with the anti-CD20 antibodyrituximab either alone or in combination with chemotherapy, such ascyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP);bendamustine (R-bendamustine), and cyclophosphamide, vincristine, andprednisone (R-CVP). Patients who relapsed after treatment with rituximabare categorized as rituximab refractory (RR) or rituximab sensitive(RS). Patients are considered RR if they progress while receivingrituximab or within 6 months of their last rituximab treatment. Patientsare considered RS if they responded to prior rituximab containingregimens and relapse more than 6 months from their last dose ofrituximab. For RS patients, approximately 40% of patients will respondto retreatment with rituximab. Clinical trial-based response andsurvival data for RR patients retreated with rituximab alone has notbeen reported, but reasonable estimates are a low response rate tosingle agent rituximab (<5%) retreatment.

Though most patients initially respond to treatment, many patients willeventually relapse and require further treatment. Furthermore, somepatients do not respond to initial therapy. More effective treatmentsare still needed for CD20-positive NHL.

In general, the overall goals of the NHL vaccine treatment are tomaximize ICD and augment and maintain the innate and adaptive immuneresponses against cancer cells. The rationale for the selection ofagents is summarized in Table 4 in which (a) Capecitabine is metabolizedto 5-FU; and (b) Leucovorin potentiates the activity of 5-FU.

Enhancing Mitigating Inducing and Conditioning Innate MaintainingImmunosuppression Coordinating Dendritic Immune Immune Agent in the TMEICD Signals and T Cells Responses Responses ALT-803 X X Avelumab XBevacizumab X X Capecitabine^(i)) X X Cyclophosphamide X X ETBX-061 X5-FU/leucovorin^(ii)) X X haNK cells X Nab-paclitaxel X X Omega-3-acidethyl esters X Oxaliplatin X Rituximab X SBRT X X

FIG. 17 exemplarily and schematically depicts the mechanism(s) by whicheach agent impacts the immune system, consequently leading to ICD. Bycombining agents that simultaneously (or sequentially) target distinctbut complementary mechanisms that enable tumor growth, the treatmentregimen aims to maximize anticancer activity and prolong the duration ofresponse to treatment.

To that end, contemplated NHL treatments combine LDMC, rituximab,bevacizumab, a cancer vaccine, low-dose radiation therapy, an IL-15superagonist, NK cell therapy, and a checkpoint inhibitor. The overallgoals of the treatment regimen are to maximize ICD and augment andmaintain the innate and adaptive immune responses against cancer cells.Specifically, the treatment is designed to interrupt the escape phase ofimmunoediting by: (a) Mitigating immunosuppression in the TME. LDMC willbe used to reduce the density of Tregs, MDSCs, and M2 macrophagescontributing to immunosuppression in the TME. Bevacizumab will be usedto cause morphological changes in the TME to promote lymphocytetrafficking; (b) Inducing and coordinating ICD signals. LDMC andlow-dose radiation therapy will be used to increase the antigenicity oftumor cells. Bevacizumab will be used to alter the TME, which allows formore efficient antigen-specific T-cell responses and makes tumor cellsmore susceptible to ICD. Omega-3-acid ethyl esters enhance ICD withoutincreasing toxicity; (c) Conditioning dendritic and T cells. A cancervaccine and an IL-15 superagonist will be used to enhance tumor-specificcytotoxic T-cell responses; (d) Enhancing innate immune responses. NKcell therapy will be used to augment the innate immune system. An IL-15superagonist will be used to enhance the activity of endogenous andintroduced NK cells. Hypofractionated-dose radiation therapy will beused to upregulate tumor cell NK ligands to enhance tumor cytotoxicityof NK cells; and (e) Maintaining immune responses. A checkpointinhibitor will be used to promote long-term anticancer immune responses.

The NHL vaccine treatment will be conducted in 2 phases: an inductionphase and a maintenance phase. The purpose of the induction phase is tostimulate immune responses against tumor cells and mitigateimmunosuppression in the TME. The purpose of the maintenance phase is tosustain ongoing immune system activity against tumor cells, creatingdurable treatment responses. Exemplary use and timing of administrationof contemplated compounds and compositions for the induction phase andthe maintenance phase are shown in FIG. 18 and FIG. 19, respectively.Therefore, the following agents and compositions are preferably used forthe induction and maintenance phases:

1. ALT-803, recombinant human super agonist interleukin-15 (IL-15)complex (also known as IL 15N72D:IL-15RαSu/IgG1 Fc complex); 2. Avelumab(BAVENCIO® injection, for IV use); 3. Bevacizumab (AVASTIN® solution forIV infusion); 4. Capecitabine (XELODA® tablets, for oral use); 5.Cyclophosphamide (CYCLOPHOSPHAMIDE Capsules, for oral use); 6. ETBX-061(Ad5 [E1-, E2b-]-MUC1); 7. 5-FU (Fluorouracil Injection, for IV useonly); 8. haNK™, NK-92 [CD16.158V, ER IL-2], Suspension for IntravenousInfusion (haNK™ for Infusion); 9. Leucovorin (LEUCOVORIN Calcium forInjection, for IV or IM use); 10. Nab-paclitaxel (ABRAXANE® forInjectable Suspension [paclitaxel protein-bound particles for injectablesuspension] [albumin-bound]); 11. Omega-3-acid ethyl esters (Lovazacapsules, for oral use); 12. Oxaliplatin (ELOXATIN® injection for IVuse); 13. Rituximab (RITUXAN® injection, for IV use); 14. SBRT.

More specifically, an exemplary treatment protocol for NHL willtypically include the following steps, phases, compounds, andcompositions:

Tumor biopsies and exploratory tumor molecular profiling will beconducted at screening, at the end of the initial induction phase (8weeks after the start of treatment), and during potential prolongedinduction and maintenance phases (depending on response). Separate bloodtubes will be collected every month in the induction phase and every 2months in the maintenance phase during routine blood draws forexploratory immunology and ctDNA/ctRNA analyses.

Tumors will be assessed at screening, and tumor response will beassessed every 8 weeks during the induction phase and every 12 weeksduring the maintenance phase by computed tomography (CT), magneticresonance imaging (MRI), or positron emission tomography-computedtomography (PET CT) of target and non-target lesions in accordance withResponse Evaluation Criteria in Solid Tumors (RECIST) Version 1.1 andimmune-related response criteria (irRC).

Induction Phase: The induction phase will comprise repeated 2 weekcycles. The treatment regimen of ALT-803, an Ad5-based vaccine(ETBX-061), haNK cells, avelumab, bevacizumab, cyclophosphamide, 5FU/leucovorin, nab-paclitaxel, omega-3-acid ethyl esters, oxaliplatin,and rituximab will be repeated every 2 weeks. Concurrent SBRT will begiven during the first four 2-week cycles. Radiation will beadministered to all feasible tumor sites using SBRT.

The induction phase of the treatment will be conducted in accordancewith the following dosing regimen:

Daily:

Omega-3-acid ethyl esters (by mouth [PO] twice a day [BID] [3×1 gcapsules and 2×1 g capsules])

Day 1, every 2 weeks:

Bevacizumab (5 mg/kg IV)

Days 1-5 and 8-12, every 2 weeks:

Cyclophosphamide (50 mg PO BID).

Days 1, 3, 5, 8, 10 and 12, every 2 weeks:

5-FU (400 mg/m2 as a continuous IV infusion over 24 hours)

Leucovorin (20 mg/m2 IV bolus)

Day 1 and 8, every 2 weeks:

Nab-paclitaxel (100 mg IV)

Oxaliplatin (40 mg/m2 IV)

Day 5, 19, 33 (every 2 weeks for 3 doses then every 8 weeks thereafter):

ETBX-061 (5×10¹¹ virus particles [VP]/dose subcutaneously [SC])

Day 8, every 2 weeks:

Avelumab (10 mg/kg IV over 1 h)

Day 8, 22, 36, 50 (every 2 weeks for 4 doses):

SBRT (not to exceed 8 Gy, exact dose to be determined by the radiationoncologist)

Day 9, every 2 weeks:

Rituximab (375 mg/m2 IV)

ALT-803 (10 μg/kg SC 30 minutes prior to haNK infusion)

Day 9 and 11, every 2 weeks:

haNK (2×10⁹ cells/dose IV)

Maintenance Phase

The duration of the maintenance phase will be up to 1 year followingcompletion of the last treatment in the induction phase. The maintenancephase will comprise repeated 2-week cycles. The treatment regimen ofALT-803, an Ad5 based vaccine (ETBX 061), haNK cells, avelumab,bevacizumab, capecitabine, cyclophosphamide, nab-paclitaxel,omega-3-acid ethyl esters, and rituximab will be repeated every 2 weeks.

The maintenance phase of the treatment will be conducted in accordancewith the following dosing regimen:

Daily:

Omega-3-acid ethyl esters (PO BID [3×1 g capsules and 2×1 g capsules])

Day 1, every 2 weeks:

Bevacizumab (5 mg/kg IV)

Nab-paclitaxel (100 mg IV)

Avelumab (10 mg/kg IV over 1 h)

Days 1-5 and 8-12, every 2 weeks:

Cyclophosphamide (50 mg PO BID)

Capecitabine (650 mg/m2 PO BID)

Day 2, every 2 weeks:

Rituximab (375 mg/m2 IV)

ALT-803 (10 μg/kg SC 30 minutes prior to haNK infusion)

haNK (2×10⁹ cells/dose IV)

Day 5, every 8 weeks thereafter:

ETBX-061 (5×10¹¹ VP/dose SC)

FIG. 20 schematically illustrates the exemplary treatment method.

Tumor Molecular Profiling: Genomic sequencing of tumor cells from tissuerelative to non-tumor cells from whole blood will be conducted toidentify tumor-specific genomic variances that may contribute to diseaseprogression and/or response to treatment. RNA sequencing will beconducted to provide expression data and give relevance to DNAmutations. Quantitative proteomics analysis will be conducted todetermine the absolute amounts of specific proteins, to confirmexpression of genes that are correlative of disease progression and/orresponse, and to determine cutoff values for response. All genomic,transcriptomic, and proteomic molecular analyses will be exploratory.Tumor molecular profiling will be performed on FFPE tumor tissue andwhole blood (subject-matched normal comparator against the tumor tissue)by next-generation sequencing and mass spectrometry-based quantitativeproteomics. Collection of tumor tissue and whole blood at screening andat the end of the initial induction phase (8 weeks after the start oftreatment) is contemplated for this treatment.

Follow-up analyses/Sample collection and Analysis: Tumor tissue andwhole blood samples will be collected and shipped in accordance with theinstruction cards included in the Tissue Specimen Kit and Blood SpecimenKit. An FFPE tumor tissue specimen is typically required for theextraction of tumor DNA, tumor RNA, and tumor protein. A whole bloodsample is typically required for the extraction of subject normal DNA.Tumor tissue and whole blood will be processed in CLIA-certified andCAP-accredited clinical laboratories.

Exploratory immunological analyses: One aim of immunotherapy treatmentis to generate antigen-specific antitumor immune responses. Exploratoryimmunology analysis will be used to provide a preliminary assessment ofimmune responses induced by the the treatments. Blood samples for immuneanalysis will be collected from subjects at screening and every month inthe induction phase and every 2 months in the maintenance phase duringroutine blood draws. PBMCs isolated by Ficoll-Hypaque density gradientseparation will be analyzed for antigen-specific immune responses usingELISpot assays for IFN-γ or granzyme B secretion after exposure to MUC1.Flow cytometry will be utilized to assess T-cell responses usingintracellular cytokine staining assay for IFN-γ or TNF-α expressionafter exposure to the tumor-associated antigen peptide, MUC1. Flowcytometry analysis for the expression of CD107a on cells will beutilized to test for degranulating cells such as CD8+ T cells and NKcells. PBMCs will be stimulated in vitro with overlapping 15-mer peptidepools encoding MUC1. Control peptide pools will involve the use ofirrelevant antigen peptide pools as a negative control and CEFT peptidemix as a positive control. CEFT is a mixture of peptides ofcytomegalovirus, EBV, influenza, and tetanus toxin. Post-stimulationanalyses of CD4 and CD8 T cells will involve the production of IFN-γ,TNF-α, and CD107a expression. Sera will be analyzed for antibodiesdirected to MUC1, neutralizing antibody titer to adenovirus (serotype5), and for potential antibody development against theIL-15N72D:IL-15RαSu/IgG1 Fc complex.

Circulating Tumor DNA and RNA Assays: Tumors evolve during therapy, anddrug-resistant cells emerge, which are difficult to detect and may causethe tumor to become resistant to the initial treatment. Blood-basedtesting for ctDNA and ctRNA can track the emergence of drug-resistanttumor cells and can identify new drug targets and treatment options forpatients. Whole blood will be collected at screening and every month inthe induction phase and every 2 months in the maintenance phase duringroutine blood draws for the analysis of ctDNA and ctRNA. Expressionlevels of specific tumor- and immune-related analytes in ctDNA and ctRNAwill be measured by qPCR and analyzed for correlations with subjectoutcomes.

Non-Small Cell Lung Cancer:

Lung cancer is the leading cause of cancer worldwide and is responsiblefor roughly 1 in 5 cancer deaths, totaling approximately 1.59 millionannual deaths. The primary risk factor for all types of lung cancer issmoking, and roughly 85-90% of lung cancer cases can be attributed tothis cause. Smoking cessation efforts have led to declining rates oflung cancer in the US over the last 25 years. Nonetheless, lung cancercontinues to impose a tremendous health burden. In the US, an estimated224,000 new cases of lung cancer where diagnosed in 2016, and roughly158,000 deaths attributable to lung cancers occurred.

Lung cancers can be histologically classified into small cell lungcancer and NSCLC. NSCLC is an umbrella category, encompassing any lungcancer that is not small cell lung cancer, which is thought to arisefrom neuroendocrine cells in the lung. NSCLC comprises roughly 85% oflung cancers, and the most common types of NSCLC include squamous cellcarcinoma, adenocarcinoma, and large cell carcinoma.

For patients with early stage, localized, and resectable disease,surgical approaches provide the best prognosis. Standard of care (SoC)surgical approaches have been reported to result in 5-year disease-freeprogression rates of roughly 70% in patients with stage 1 NSCLC.However, this applies to only a small minority of patients, as 70% ofnewly diagnosed lung cancer patients present with advanced stagedisease, and most of these patients have metastatic disease. Surgery isnot recommended for most patients with stage 3 or 4 NSCLC.

In general, the overall goals of the NSCLC vaccine treatment presentedherein are to maximize ICD and augment and maintain the innate andadaptive immune responses against cancer cells. The rationale for theselection of agents included in this treatment is summarized in Table 5in which (i) denotes tumor molecular profiling will determine whetherETBX-021 will be administered; (ii) denotes tumor molecular profilingwill determine whether GI-4000 will be administered; (iii) denotescapecitabine is metabolized to 5-FU; (iv) denotes cisplatin will beadministered to subjects with the squamous cell carcinoma subtype.Oxaliplatin will be administered to subjects with the adenocarcinomasubtype; (v) denotes Leucovorin potentiates the activity of 5-FU, and(vi) denotes that either nivolumab or avelumab will be administered.

Enhancing Mitigating Inducing and Conditioning Innate MaintainingImmunosuppression Coordinating Dendritic and Immune Immune Agent in theTME ICD Signals T Cells Responses Responses Non-Marketed productsALT-803 X X ETBX-011 X ETBX-021^(i)) X ETBX-051 X ETBX-061 XGI-4000^(ii)) X GI-6207 X GI-6301 X haNK cells X Approved productsBevacizumab X X Capecitabine^(iii)) X X Cisplatin/oxaliplatin^(iv)) XCyclophosphamide X X 5-FU/leucovorin^(v)) X X Fulvestrant XNab-paclitaxel X X Nivolumab/avelumab^(vi)) X Omega-3-acid ethyl estersX SBRT X X

FIG. 21 depicts the mechanism(s) by which each agent impacts the immunesystem, consequently leading to ICD. By combining agents thatsimultaneously target distinct but complementary mechanisms that enabletumor growth, the treatment regimen aims to maximize anticancer activityand prolong the duration of response to treatment.

To that end, contemplated NSCLC treatments combine LDMC, bevacizumab,cancer vaccines, low-dose radiation therapy, an IL-15 superagonist, NKcell therapy, and a checkpoint inhibitor. The overall goals of thetreatment regimen are to maximize ICD and augment and maintain theinnate and adaptive immune responses against cancer cells. Specifically,the treatment is set up to interrupt the escape phase of immunoeditingby: (a) Mitigating immunosuppression in the TME. LDMC will be used toreduce the density of Tregs, MDSCs, and M2 macrophages contributing toimmunosuppression in the TME. Bevacizumab will be used to causemorphological changes in the TME to promote lymphocyte trafficking; (b)Inducing and coordinating ICD signals. LDMC and low-dose radiationtherapy will be used to increase the antigenicity of tumor cells.Bevacizumab will be used to alter the TME, which allows for moreefficient antigen-specific T-cell responses and makes tumor cells moresusceptible to ICD. Fulvestrant will be used to enhance ADCC andcytotoxic T-cell activity. Omega-3-acid ethyl esters enhances ICDwithout increasing toxicity; (c) Conditioning dendritic and T cells. Acancer vaccine and an IL-15 superagonist will be used to enhancetumor-specific cytotoxic T-cell responses; (d) Enhancing innate immuneresponses. NK cell therapy will be used to augment the innate immunesystem. An IL-15 superagonist will be used to enhance the activity ofendogenous and introduced NK cells. Hypofractionated low-dose radiationtherapy will be used to upregulate tumor cell NK ligands to enhancetumor cytotoxicity of NK cells; and (e) Maintaining immune responses. Acheckpoint inhibitor will be used to promote long-term anticancer immuneresponses.

The NSCLC vaccine treatment will be conducted in 2 phases: an inductionphase and a maintenance phase. The purpose of the induction phase is tostimulate immune responses against tumor cells and mitigateimmunosuppression in the TME. The purpose of the maintenance phase is tosustain ongoing immune system activity against tumor cells, creatingdurable treatment responses. Exemplary use and timing of administrationof contemplated compounds and compositions for the induction phase andthe maintenance phase are shown in FIG. 22 and FIG. 23, respectively.Therefore, the following agents and compositions are preferably used forthe induction and maintenance phases:

1. ALT-803, recombinant human super agonist IL-15 complex (also known asIL 15N72D:IL-15RαSu/IgG1 Fc complex); 2. ETBX-011 (Ad5 [E1-, E2b-]-CEA);3. ETBX-021 (Ad5 [E1-, E2b-]-HER2); 4. ETBX-051 (Ad5 [E1-,E2b-]-Brachyury); 5. ETBX-061 (Ad5 [E1-, E2b-]-MUC1); 6. GI-4000 (Rasyeast vaccine); 7. GI-6207 (CEA yeast vaccine); 8. GI-6301 (Brachyuryyeast vaccine); 9. haNK™, NK-92 [CD16.158V, ER IL-2], Suspension for IVInfusion (haNK™ for Infusion); 10. Avelumab (BAVENCIO® injection, for IVuse); 11. Bevacizumab (AVASTIN® solution for IV infusion); 12.Capecitabine (XELODA® tablets, for oral use); 13. Cisplatin (CISplatininjection); 14. Cyclophosphamide (CYCLOPHOSPHAMIDE Capsules, for oraluse); 15. 5-FU (Fluorouracil Injection, for IV use only); 16.Fulvestrant (FASLODEX® for injection); 17. Leucovorin (LEUCOVORINCalcium for Injection, for IV or IM use); 18. Nab-paclitaxel (ABRAXANE®for Injectable Suspension [paclitaxel protein-bound particles forinjectable suspension] [albumin-bound]); 19. Nivolumab (OPDIVO®injection, for IV use); 20. Omega-3-acid ethyl esters (Lovaza capsules,for oral use); 21. Oxaliplatin (ELOXATIN® injection for IV use); and 22.SBRT.

More specifically, an exemplary treatment protocol for NSCLC willtypically include the following steps, phases, compounds, andcompositions:

Tumors will be assessed at screening, and tumor response will beassessed every 8 weeks during the induction phase and every 12 weeksduring the maintenance phase by computed tomography (CT), magneticresonance imaging (MRI), or positron emission tomography (PET)-CT oftarget and non-target lesions in accordance with Response EvaluationCriteria in Solid Tumors (RECIST) Version 1.1 and immune-relatedresponse criteria (irRC).

Prospective Tumor Molecular Profiling: Prospective tumor molecularprofiling will be conducted to inform HER2 expression and Ras mutationalstatus and will be used to determine whether ETBX-021 and GI-4000 willbe administered. All subjects will receive ETBX-011, ETBX-051, ETBX-061,GI-6207, and GI-6300 regardless of their tumor molecular profile.Prospective tumor molecular profiling will be performed on FFPE tumortissue and whole blood (subject-matched normal comparator against thetumor tissue) collected at screening.

Subjects will receive ETBX-021 if their tumor overexpresses HER2 (≥750attomole/μg of tumor tissue, as determined by quantitative proteomicswith mass spectrometry). Subjects will receive GI-4000 if their tumor ispositive for specific Ras mutations, as determined by whole genomesequencing. GI-4000 is 4 separate products from the GI-4000 series(GI-4014, GI-4015, GI-4016, and GI-4020); each of these expresses acombination of mutated Ras oncoproteins. The specific Ras mutation willdetermine which GI-4000 product will be used for treatment (GI-4014 forG12V, GI-4015 for G12C, GI-4016 for G12D, GI-4020 for G12R or Q61H, andGI-4014, GI-4015, or GI-4016 for Q61L or Q61R).

Induction Phase: The induction phase will comprise repeated 2-weekcycles for a maximum treatment period of 1 year. The treatment regimenof omega-3-acid ethyl esters, cyclophosphamide, cisplatin oroxaliplatin, 5 FU/leucovorin, nab-paclitaxel, bevacizumab, ALT-803, haNKcells, Ad5-based vaccines (ETBX-011, ETBX-021, ETBX-051, and ETBX-061),yeast-based vaccines (GI-4000, GI-6207, and GI-6301), nivolumab oravelumab, fulvestrant, and radiation therapy will be repeated every 2weeks. Concurrent SBRT will be given during the first four 2-weekcycles. Radiation will be administered to all feasible tumor sites usingSBRT. An exemplary induction phase of NSCLC treatment will be conductedin accordance with the following dosing regimen:

Daily:

Omega-3-acid ethyl esters (by mouth [PO] BID [3×1 g capsules and 2×1 gcapsules])

Day 1, every 2 weeks:

Bevacizumab (5 mg/kg IV)

Day 1, every 4 weeks (every other treatment cycle):

Fulvestrant (500 mg IM)

Days 1-5 and 8-12, every 2 weeks:

Cyclophosphamide (50 mg PO twice a day [BID]).

Days 1, 3, 5, 8, 10 and 12, every 2 weeks:

5-FU (400 mg/m2 continuous IV infusion over 24 hours)

Leucovorin (20 mg/m2 IV bolus)

Day 1 and 8, every 2 weeks:

Nab-paclitaxel (100 mg IV)

Cisplatin (40 mg/m2 IV) or oxaliplatin (40 mg/m2 IV)

Cisplatin will be administered to subjects with the squamous cellcarcinoma subtype. Oxaliplatin will be administered to subjects with theadenocarcinoma subtype.

Day 5, 19, 33 (every 2 weeks for 3 doses then every 8 weeks thereafter):

ETBX-011, ETBX-021, ETBX-051, ETBX-061 (5×10¹¹ virus particles[VI:]/vaccine/dose subcutaneously [SC])

GI-4000, GI-6207, GI-6301, (40 yeast units [YU]/vaccine/dose SC), 2hours after administration of the Ad5-based vaccines

Prospective tumor molecular profiling will determine whether ETBX-021and GI-4000 will be administered, as described above.

Day 8, every 2 weeks:

Nivolumab (3 mg/kg IV over 1 hour) or avelumab (10 mg/kg IV over 1 hour)

Day 8, 22, 36, 50 (every 2 weeks for 4 doses):

SBRT (not to exceed 8 Gy, exact dose to be determined by the radiationoncologist)

Day 9, every 2 weeks:

ALT-803 (10 μg/kg SC 30 minutes prior to haNK infusion)

Day 9 and 11, every 2 weeks:

haNK (2×10⁹ cells/dose IV)

Maintenance Phase: The duration of the maintenance phase will be up to 1year following completion of the last treatment in the induction phase.The maintenance phase will comprise repeated 2-week cycles. Thetreatment regimen of omega-3-acid ethyl esters, cyclophosphamide,capecitabine, nab-paclitaxel, bevacizumab, ALT-803, haNK cells,Ad5-based vaccines (ETBX-011, ETBX-021, ETBX-051, and ETBX-061),yeast-based vaccines (GI-4000, GI-6207, and GI-6301), nivolumab oravelumab, and fulvestrant will be repeated every 2 weeks. An exemplarymaintenance phase of the treatment will be conducted in accordance withthe following dosing regimen:

Daily:

Omega-3-acid ethyl esters (PO BID [3×1 g capsules and 2×1 g capsules])

Day 1, every 2 weeks:

Bevacizumab (5 mg/kg IV)

Nab-paclitaxel (100 mg IV)

Nivolumab (3 mg/kg IV over 1 hour) or avelumab (10 mg/kg IV over 1 hour)

Day 1, every 4 weeks (every other treatment cycle):

Fulvestrant (500 mg IM)

Days 1-5 and 8-12, every 2 weeks:

Capecitabine (650 mg/m2 PO BID)

Cyclophosphamide (50 mg PO BID)

Day 2, every 2 weeks:

ALT-803 (10 μg/kg SC) (30 minutes prior to haNK infusion)

haNK (2×10⁹ cells/dose IV)

Day 5, every 8 weeks thereafter:

ETBX-011, ETBX-021, ETBX-051, ETBX-061 (5×10¹¹ VP/vaccine/dose SC)

GI-4000, GI-6207, GI-6301 (40 YU/vaccine/dose SC), 2 hours afteradministration of the Ad5 based vaccines.

Prospective molecular profiling will determine whether ETBX-021 andGI-6207 will be administered, as described above. FIG. 24 schematicallyillustrates the exemplary treatment protocol.

Tumor Molecular Profiling: Genomic sequencing of tumor cells from tissuerelative to non-tumor cells from whole blood will be conducted toidentify tumor-specific genomic variances that may contribute to diseaseprogression and/or response to treatment. RNA sequencing will beconducted to provide expression data and give relevance to DNAmutations. Quantitative proteomics analysis will be conducted todetermine the absolute amounts of specific proteins, to confirmexpression of genes that are correlative of disease progression and/orresponse, and to determine cutoff values for response. All genomic,transcriptomic, and proteomic molecular analyses will be exploratory,except for the prospective tumor molecular analysis of HER2 expressionby quantitative proteomics and analysis of Ras mutational status bygenomic sequencing to determine whether ETBX-021 and GI-4000 will beadministered.

Follow-up analyses/Sample Collection and Analysis: Tumor molecularprofiling will be performed on FFPE tumor tissue and whole blood(subject-matched normal comparator against the tumor tissue) bynext-generation sequencing and mass spectrometry-based quantitativeproteomics. Collection of tumor tissue and whole blood at screening andat the end of the initial induction phase (8 weeks after the start oftreatment) is contemplated for this treatment. An FFPE tumor tissuespecimen is typically required for the extraction of tumor DNA, tumorRNA, and tumor protein. A whole blood sample is typically required forthe extraction of subject normal DNA. Tumor tissue and whole blood willbe processed in CLIA-certified and CAP-accredited clinical laboratories.

Blood samples for immune analysis will be collected from subjects atscreening and every month in the induction phase and every 2 months inthe maintenance phase during routine blood draws. PBMCs isolated byFicoll-Hypaque density gradient separation will be analyzed forantigen-specific immune responses using ELISpot assays for IFN-γ orgranzyme B secretion after exposure to the following tumor-associatedantigen peptides: CEA, Brachyury, and MUC1, and if ETBX-021 and GI-4000are administered, HER2 and mutant Ras, respectively. Flow cytometry willbe utilized to assess T-cell responses using intracellular cytokinestaining assay for IFN-γ or TNF-α expression after exposure to thetumor-associated antigen peptides. Flow cytometry analysis for theexpression of CD107a on cells will be utilized to test for degranulatingcells such as CD8+ T cells and NK cells. PBMCs will be stimulated invitro with overlapping 15-mer peptide pools encoding thetumor-associated antigens mentioned above. Control peptide pools willinvolve the use of irrelevant antigen peptide pools as a negativecontrol and CEFT peptide mix as a positive control. CEFT is a mixture ofpeptides of CMV, Epstein-Barr virus, influenza, and tetanus toxin.Post-stimulation analyses of CD4+ and CD8+ T cells will involve theproduction of IFN-γ, TNF-α, and CD107a expression. Sera will be analyzedfor antibodies directed to the aforementioned tumor-associated antigens,neutralizing antibody titer to adenovirus (serotype 5), and forpotential antibody development against the IL-15N72D:IL-15RαSu/IgG1 Fccomplex.

Circulating Tumor DNA and RNA Assays: Tumors evolve during therapy, anddrug-resistant cells emerge, which are difficult to detect and may causethe tumor to become resistant to the initial treatment. Blood-basedtesting for ctDNA and ctRNA can track the emergence of drug-resistanttumor cells and can identify new drug targets and treatment options forpatients. Whole blood will be collected at screening and every month inthe induction phase and every 2 months in the maintenance phase duringroutine blood draws for the analysis of ctDNA and ctRNA. Expressionlevels of specific tumor- and immune-related analytes in ctDNA and ctRNAwill be measured by qPCR and analyzed for correlations with subjectoutcomes.

Pancreatic Cancer:

Pancreatic cancer is projected to be the second leading cause ofcancer-related death in the US, with an estimated 43,090 deaths from thedisease and an estimated 53,670 new cases expected in 2017. It is the12^(th) most common cancer worldwide, with around 338,000 new casesdiagnosed in 2012 (2% of the total). The prognosis is poor, and as aresult, pancreatic cancer is the 7^(th) most common cause of cancerdeath worldwide, with more than 330,000 deaths from pancreatic cancer in2012 (4% of the total).

The pancreas is composed of 2 main cell types, exocrine and endocrine.Exocrine cells produce digestive enzymes, while the endocrine cells ofthe islets of Langerhans produce the hormones insulin and glucagon.Endocrine tumors typically have a better prognosis but only account for6% of the pancreatic cancer that develops. Exocrine tumors, on the otherhand, are rarely curable and are by far the most common type ofpancreatic cancer, with adenocarcinoma accounting for about 94% ofcancers of the exocrine pancreas. Incidence rates for pancreatic cancerhave increased by approximately 1% per year from 2004 to 2013 in whiteindividuals, but have remained the same for black individuals.

The prognosis for patients with pancreatic adenocarcinoma is very poor,with an overall median survival of 5 to 8 months; fewer than 5% ofpatients live for more than 5 years. Surgical resection of thepancreatic cancer and subsequent adjuvant chemotherapy is the maintreatment option required to achieve long-term survival. It can beachieved in about 15% to 20% of newly diagnosed patients; however,recurrence is common, even in cases where optimal resection is achieved.For the majority of the patients who present with more advanced disease,treatment typically comprises chemotherapy alone or supportive care formetastatic patients, and chemotherapy with or without radiation forthose with locally advanced disease. The prognosis for these patients iseven less promising, with a 5-year survival of 2%.

A majority of patients with pancreatic cancer present with advanceddisease. Survival rates for this group are remarkably low, with just 2%of patients with metastatic disease surviving 5 years from the time ofdiagnosis. A small group of patients (9%) are diagnosed with localizedresectable disease; however, even for this group, 5-year survival ratesare poor, at just over 25%. Standard of care treatment for patients withpancreatic cancer is treatment with FOLFIRINOX, which improves OS andPFS over monotherapy with gemcitabine; however, FOLFIRINOX is availableonly to patients in relatively good health (ECOG 0 or 1), and prognosisfor patients receiving treatment remains grim, with median PFS of 6.4months and median OS of 11.1 months (Conroy 2011). Novel treatmentoptions that can produce long-lasting, durable responses in asubstantial fraction of patients are clearly needed for patients withpancreatic cancer.

In general, the overall goals of the PANC vaccine treatment presentedherein are to maximize immunological cell death (ICD) while maintainingand augmenting patients' antitumor adaptive and innate response tocancers. The rationale for the selection of agents included in thetreatment is summarized in Table 6.

Induction and Coordination Maintenance Overcoming of Dendritic Enhancingof the suppressive Immunogenic and T Cells NK Cell Immune Agent TMESignals Conditioning Responses Responses Cyclophosphamide X OxaliplatinX 5-FU/capecitabine X Nab-paclitaxel X X Bevacizumab X X Avelumab XRadiation therapy X ALT-803 X X aNK for Infusion X Ad5 vaccine X GI-4000RAS vaccine X

FIG. 25 depicts the mechanism(s) by which each agent impacts the immunesystem, consequently leading to ICD. By combining agents thatsimultaneously target distinct but complementary mechanisms that enabletumor growth, the treatment regimen aims to maximize anticancer activityand prolong the duration of response to treatment.

To that end, contemplated PANC treatments are set up to achieve thespecific and complementary aims of: 1) overcoming the suppressive TME;2) molecularly-informed induction of immunogenic signals; 3) dendriticand T cell conditioning; 4) NK cell transplant; and 5) maintenance ofthe immune response and induction of durable long-term remission throughadministration of LDMC.

The PANC vaccine treatment will be conducted in 2 phases: an inductionphase and a maintenance phase. The purpose of the induction phase is tostimulate immune responses against tumor cells and mitigateimmunosuppression in the TME. The purpose of the maintenance phase is tosustain ongoing immune system activity against tumor cells, creatingdurable treatment responses. Exemplary use and timing of administrationof contemplated compounds and compositions for the induction phase andthe maintenance phase are shown in FIG. 26 and FIG. 27, respectively.Therefore, the following agents and compositions are preferably used forthe induction and maintenance phases:

1. CYCLOPHOSPHAMIDE tablets, for oral use; 2. ELOXATIN® (oxaliplatin forinjection, USP); 3. XELODA (capecitabine) tablets, for oral use; 4.Fluorouracil Injection, for intravenous use; 5. LEUCOVORIN Calcium forInjection, for IV or IM use; 6. ABRAXANE® (nab-paclitaxel); 7. AVASTIN(bevacizumab); 8. ALT-803, recombinant human super agonistinterleukin-15 (IL-15) complex (also known as IL 15N72D:IL-15RαSu/IgG1Fc complex); 9. aNK™, NK-92 [CD16.158V, ER IL-2] (high-affinityactivated natural killer cell line, [aNK™ for Infusion]); 10. ETBX-011:Ad5 [E1-, E2b-]-CEA (carcinoembryonic antigen); 11. Avelumab, a humananti-PD-L1 IgG1 monoclonal antibody; 12. GI-4000, a vaccine derived fromrecombinant Saccharomyces cerevisiae yeast expressing mutant Rasproteins.

More specifically, an exemplary treatment protocol for PANC willtypically include the following steps, phases, compounds, andcompositions:

Tumor biopsies and tumor molecular profiling will be conducted atscreening and at the end of the initial induction (8 weeks) and during apotential prolonged induction phase (depending on response). Inaddition, during routine weekly blood draws, a separate blood tube willbe collected to analyze blood for changes in circulating RNA. Tumorswill be assessed at screening, and tumor response will be assessed every8 weeks during the induction phase, and every 3 months during themaintenance phase by computed tomography (CT), magnetic resonanceimaging (MRD, or positron emission tomography (PET) of target andnon-target lesions according to Response Evaluation Criteria in SolidTumors (RECIST) Version 1.1 and immune-related response criteria (irRC).

Induction Phase: The induction phase will comprise repeated 2 weekcycles of low-dose radiation and metronomic chemotherapy. The treatmentregimen of cyclophosphamide, oxaliplatin, 5-FU/leucovorin,nab-paclitaxel, bevacizumab, ALT-803, aNK, vaccines (Ad5 and GI-4000),and avelumab will be repeated every 2 weeks. Concurrent stereotacticbody radiotherapy (SBRT) will be given during the first four 2-weekcycles. Radiation will be administered to all feasible tumor sites usingSBRT. Techniques contemplated include linear-accelerator based therapies(3D and intensity-modulated radiation therapy [IMRT]) and gamma andcyber knife.

The induction treatment will continue until the subject experiences PDor unacceptable toxicity (not correctable with dose reduction). Subjectsthat have a CR in the induction phase will enter the maintenance phaseof the treatment. Response assessments using CT/MRI evaluated accordingto RECIST Version 1.1 and irRC will be performed every 8 weeks duringthe induction phase.

Days 1-5 and 8-12, every 2 weeks:

Cyclophosphamide (50 mg twice a day [BID]).

Day 1 and 8, every 2 weeks:

Oxaliplatin (40 mg/m2 IV)

Nab-paclitaxel (125 mg IV)

Day 1 every 2 weeks:

Bevacizumab (5 mg/kg IV)

Days 1, 3, 5, 8, 10 and 12, every 2 weeks:

5-fluorouracil (400 mg/m2 over 24 hours as a continuous infusion)

Leucovorin (20 mg/m2 IV bolus)

Day 8, 22, 36, 50 (every 2 weeks for 4 doses):

SBRT (8 Gy)

Day 9, every 2 weeks:

ALT-803 (10 μg/kg subcutaneously [SC] 30 minutes prior to aNK infusion)

Day 9 and 11, every 2 weeks:

aNK (2×10⁹ cells/dose IV)

Day 5, 19, 33 (every 2 weeks for 3 doses then every 8 weeks thereafter):

Ad5 [E1-, E2b-]-CEA (5×10¹¹ VP/dose SC)

GI-4000 (40 yeast units [YU] SC; use dependent on genomic sequencingindicating required KRAS mutations)

Day 8, every 2 weeks:

Avelumab (10 mg/kg IV over 1 h)

Maintenance Phase: The duration of the maintenance phase will be 1 yearfollowing completion of the last treatment in the induction phase.Treatment will continue throughout the maintenance phase unless thesubject experiences PD or unacceptable toxicity. Response assessmentsusing CT/MRI evaluated according to RECIST Version 1.1 and irRC will beperformed every 3 months during the maintenance phase.

Days 1-5 and 8-12, every 2 weeks:

Cyclophosphamide (50 mg BID)

Capecitabine (650 mg/m2 PO BID)

Day 1, every 2 weeks:

Nab-paclitaxel (125 mg IV)

Bevacizumab (5 mg/kg IV)

Avelumab (10 mg/kg IV over 1 h)

Day 2, every 2 weeks:

ALT-803 (10 μg/kg SC) (30 minutes prior to aNK infusion)

aNK (2×10⁹ cells/dose IV)

Day 5, every 8 weeks thereafter:

Ad5 [E1-, E2b-]-CEA (5×10¹¹ VP/dose SC)

GI-4000 (40 YU SC)

FIG. 28 schematically illustrates the exemplary treatment protocol.

Follow-up analyses/sample collection and analysis: Exploratory genomics,transcriptomics, circulating RNA and proteomics molecular profiling willbe performed on FFPE tumor tissue and whole blood (subject matchednormal comparator against the tumor tissue) by next-generationsequencing and mass spectrometry-based quantitative proteomics. Duringthe induction phase, blood samples will be collected on a weekly basisfor molecular profiling. During the maintenance phase, blood sampleswill be collected on a monthly basis for molecular profiling; a sampleof 22.5 mL is required at each blood draw.

Sample Collection and Analysis for cell free DNA and cell free RNA: Thespecimens are 10 mL of whole blood drawn into Cell-free RNA BCT® tubesor Cell-free DNA BCT® tubes containing RNA or DNA stabilizers,respectively. CtRNA is stable in whole blood in the Cell-free RNA BCTtubes for 7 days; ctDNA is stable in whole blood in the Cell-free DNABCT Tubes for 14 days. These nucleic acid stabilizers allow time forshipping of patient samples without degradation of ctRNA or ctDNA. Wholeblood in 10 mL tubes is centrifuged to fractionate plasma at 1600 rcffor 20 minutes. The plasma is separated and centrifuged at 16,000 rcffor 10 minutes to remove cell debris. CtDNA and ctRNA were extractedfrom 2 mL of plasma with a proprietary in-house developed protocol usingQiagen reagents. The protocol was designed to remove potentialcontaminating blood cells, other impurities and maintain stability ofthe nucleic acids during the extraction. All nucleic acids are kept inbar-coded matrix storage tubes. DNA is stored at −4° C. and RNA isstored at −80° C. or reverse-transcribed to complementary DNA (cDNA) andcDNA is stored at −4° C.

Expression of PD-L1 is measured by quantitative real-time PCR of ct-cDNAusing primers specific for this gene. Amplification is performed in a 10μl reaction mix containing 2 μL cDNA, the primer and probe. (3-actin isused as an internal control for the input level of ct-cDNA. A standardcurve of samples with known concentrations of PD-L1 is run on each PCRplate as well as positive and negative controls for each gene. Testsamples are identified by scanning the 2D barcode on the matrix tubescontaining the nucleic acids. Delta Ct (dCT) is calculated from the Ctvalue of PD-L1 subtracted by the Ct value of (3-actin. Relativeexpression of patient specimens is calculated using a standard curve ofdelta Cts of serial dilutions of Universal Human Reference RNA set at agene expression value of 10 (when the delta CTs are plotted against thelog concentration of PD-L1). The PD-L1 levels will be analyzed with theprimary and secondary outcomes to identify statistically and clinicallysignificant correlations.

Immunology Analysis: Blood samples for immune analysis will be collectedfrom subjects prior to their first treatment and again at Day 1 of eachtreatment cycle and at the end of the treatment. Pre- and post-therapyPBMCs, isolated by Ficoll-Hypaque density gradient separation, will beanalyzed for antigen-specific immune responses using ELISpot assays forIFN-γ or granzyme B secretion after exposure to CEA peptides. Flowcytometry will be utilized to assess T cell responses usingintracellular cytokine staining assay for IFN-γ or TNF-α expressionafter exposure to CEA peptides. Flow cytometry analysis for theexpression of CD107a on cells will be utilized to test for degranulatingcells such as CD8+ T cells and NK cells. PBMCs will be stimulated invitro with overlapping 15-mer peptide pools encoding thetumor-associated antigen CEA. Control peptide pools will involve the useof irrelevant antigen peptide pools as a negative control and CEFTpeptide mix as a positive control. CEFT is a mixture of peptides of CMV,Epstein-Barr virus, influenza, and tetanus toxin. Post-stimulationanalyses of CD4 and CD8 T cells will involve the production of IFN-γ,TNF-α, and CD107a expression. Sera will be analyzed pre- andpost-therapy for CEA directed antibody, neutralizing antibody titer toadenovirus (serotype 5), and for potential antibody development againstthe IL-15N72D:IL-15RαSu/IgG1 Fc complex.

Soft Tissue Sarcoma:

Soft-tissue sarcomas are relatively uncommon cancers. They account forless than 1% of all new cancer cases each year. This may be becausecells in soft tissue, in contrast to tissues that more commonly giverise to malignancies, are not continuously dividing cells.

In 2006, about 9,500 new cases were diagnosed in the United States.Soft-tissue sarcomas are more commonly found in older patients (>50years old) although in children and adolescents under age 20, certainhistologies are common (rhabdomyosarcoma, synovial sarcoma).

In general, the overall goals of the soft tissue sarcome vaccinetreatment are to maximize ICD and augment and maintain the innate andadaptive immune responses against cancer cells. Similar to the treatmentcompounds and compositions above, the following agents and compositionsare preferably used for the induction and maintenance phases:

1. CYCLOPHOSPHAMIDE tablets, for oral use; 2. Trabectedin forintravenous use; 3. AVASTIN (bevacizumab) solution for IV infusion; 4.Avelumab, a human anti-PD-L1 IgG1 monoclonal antibody; 5. ABRAXANE®(nab-paclitaxel) for injectable suspension; 6. Doxorunicin; 7. ALT-803,recombinant human super agonist interleukin-15 (IL-15) complex (alsoknown as IL 15N72D:IL-15RαSu/IgG1 Fc complex); 8. HaNK™, NK-92(activated natural killer cell line, aNK™ for infusion; 9. Ad5 [E1-,E2b-]-MUC1; 10. Ad5 [E1-, E2b-]-Brachyury; and 11. GI 6301-YeastBrachyury.

More specifically, an exemplary treatment protocol for soft tissuesarcoma will typically include the following steps, phases, compounds,and compositions:

Tumor biopsies and tumor molecular profiling will be conducted atscreening and at the end of the initial induction (8 weeks) and during apotential prolonged induction phase (depending on response). Inaddition, during routine weekly blood draws, a separate blood tube willbe collected to analyze blood for changes in circulating RNA. Tumorswill be assessed at screening, and tumor response will be assessed every8 weeks during the induction phase, and every 3 months during themaintenance phase by computed tomography (CT), magnetic resonanceimaging (MRI), or positron emission tomography (PET) of target andnon-target lesions according to Response Evaluation Criteria in SolidTumors (RECIST) Version 1.1 and immune-related response criteria (irRC).

Induction Phase: The induction phase will comprise repeated 2 weekcycles of low-dose radiation and metronomic chemotherapy. The treatmentregimen of cyclophosphamide, doxorubicin, nab-paclitaxel, bevacizumab,trabectedin, ALT-803, HaNK, avelumab, vaccine, and radiation therapywill be repeated every 2 weeks. Concurrent stereotactic bodyradiotherapy (SBRT) will be given during the first four 2-week cycles.Radiation will be administered to all feasible tumor sites using SBRT.Techniques contemplated include linear-accelerator based therapies (3Dand intensity-modulated radiation therapy [IMRT]).

The induction treatment will continue until the subject experiences PDor unacceptable toxicity (not correctable with dose reduction). Subjectsthat have a CR in the induction phase will enter the maintenance phaseof the treatment. Response assessments using CT/MRI will be performedevery 8 weeks during the induction phase and will be evaluated accordingto RECIST Version 1.1 and irRC.

Days 1-5 (weekly):

Cyclophosphamide 50 mg twice a day (BID)

Day 1 (weekly):

Doxorubicin 20 mg/m2 IV

Day 1 (every 2 weeks):

Bevacizumab 5 mg/kg IV

Days 1 (weekly):

Trabectedin 0.5 mg/kg IV

nab-paclitaxel 100 mg IV

Day 8, 22, 36, 50 (every other week for 4 doses):

SBRT 8 Gy

Day 9 (every 2 weeks):

ALT-803 10 μg/kg SC

Day 9 and 11 (every 2 weeks):

HaNK 2×10⁹ cells/dose IV

Day 5, 19, 33 (every 2 weeks for 3 doses then every 8 weeks thereafter):

Ad5 [E1-, E2b-]-MUC1 Ad5 [E1-, E2b-]-Brachyury 5×10¹¹ VP/dose SC

GI-6301 Yeast Brachyury 40 YU SC

Day 8 (every 2 weeks):

Avelumab 10 mg/kg by 1 h IV

Maintenance Phase: The duration of the maintenance phase will be 1 yearfollowing completion of the last treatment in the induction phase.Treatment will continue throughout the maintenance phase unless thesubject experiences PD or unacceptable toxicity. Response assessmentsusing CT/MRI evaluated according to RECIST Version 1.1 and irRC will beperformed every 3 months during the maintenance phase.

Days 1-5 (weekly):

Cyclophosphamide 50 mg twice a day (BID)

Day 1 (every 2 weeks):

nab-paclitaxel 100 mg IV

Avelumab 10 mg/kg IV

Bevacizumab 5 mg/kg IV

Trabectedin 0.5 mg/kg IV

Day 2 (every 2 weeks):

HaNK 2×10⁹ cells/dose IV

ALT-803 10 μg/kg SC

Day 5 (every 8 weeks thereafter):

Ad5 [E1-, E2b-]-MUC1 Ad5 [E1-, E2b-]-Brachyury 5×10¹¹ VP/dose SC

GI-6301 Yeast Brachyury 40 YU SC

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.Unless the context dictates the contrary, all ranges set forth hereinshould be interpreted as being inclusive of their endpoints, andopen-ended ranges should be interpreted to include commerciallypractical values. Similarly, all lists of values should be considered asinclusive of intermediate values unless the context indicates thecontrary.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise. Furthermore, and unless the context dictatesotherwise, the term “coupled to” is intended to include both directcoupling (in which two elements that are coupled to each other contacteach other) and indirect coupling (in which at least one additionalelement is located between the two elements). Therefore, the terms“coupled to” and “coupled with” are used synonymously.

As used herein, the term “treat”, “treating” or “treatment” of anydisease or disorder refers, in one embodiment, to the administration ofone or more compounds or compositions for the purpose of amelioratingthe disease or disorder (e.g., slowing or arresting or reducing thedevelopment of the disease or at least one of the clinical symptomsthereof). In another embodiment “treat”, “treating”, or “treatment”refers to the administration of one or more compounds or compositionsfor the purpose of alleviating or ameliorating at least one physicalparameter including those which may not be discernible by the patient.In yet another embodiment, “treat”, “treating”, or “treatment” refers tothe administration of one or more compounds or compositions for thepurpose of modulating the disease or disorder, either symptomatically,(e.g., stabilization of a discernible symptom), physiologically, (e.g.,breaking the escape phase of cancer immunoediting, induction of anelimination phase of cancer immunoediting, reinstatement of equilibriumphase of cancer immunoediting), or both. In yet another embodiment,“treat”, “treating”, or “treatment” refers to the administration of oneor more compounds or compositions for the purpose of preventing ordelaying the onset or development or progression of the disease ordisorder. The terms “treat”, “treating”, and “treatment” may result, forexample in the case of cancer in the stabilization of the disease,partial, or complete response. However, and especially where the canceris treatment resistant, the terms “treat”, “treating”, and “treatment”do not imply a cure or even partial cure. As also used herein, the term“patient” refers to a human (including adults and children) or othermammal that is diagnosed or suspected to have a disease, and especiallycancer.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A method of providing a coordinated treatmentregimen for treating a tumor, comprising: reverting an escape phase ofthe tumor by administering at least a first pharmaceutical compositionthat reduces immune suppression in a tumor microenvironment; inducing anelimination phase of the tumor by administering at least a secondpharmaceutical composition that enhances at least one of an adaptiveimmune response and an innate immune response; and maintaining anequilibrium phase of the tumor by administering at least a thirdpharmaceutical composition that biases the adaptive immune responsetowards a T_(H)1 response.
 2. The method of claim 1 wherein the firstpharmaceutical composition comprises a drug that is bound to an albumin,wherein the albumin is optionally a nanoparticulate albumin.
 3. Themethod of claim 2 further comprising an antibody or fragment thereofbound to the albumin.
 4. The method of claim 2 wherein the drug isselected form the group consisting of Bendamustine, Bortezomib,Cabazitaxel, Chlorambucil, Cisplatin, Cyclophosphamide, Dasatinib,Docetaxel, Doxorubicin, Epirubicin, Erlotinib, Etoposide, Everolimus,Gefitinib, Idarubicin, Hydroxyurea, Imatinib, Lapatinib, Melphalan,Mitoxantrone, Nilotinib, Oxiplatin, Paclitaxel, Pazopanib, Pemetrexed,Rapamycin, Romidepsin, Sorafenib, Vemurafenib, Sunitinib, Teniposide,Vinblastine, Vinorelbine, and Vincristine.
 5. The method of claim 2wherein the antibody or fragment thereof is selected form the groupconsisting of Reopro, Kadcyla, Campath, Simulect, Avastin, Benlysta,Adcetris, Cimzia, Rbitux, Prolia, Zevalin, Tysabri, Gazyva, Arzerra,Xolair, Vectibix, Perjeta, Cyramza, Lucentis, Rituxan, Bexar, Yondelis,and Herceptin. 6-21. (canceled)
 22. The method of claim wherein theantibody or fragment thereof binds specifically to a component of anecrotic cell.
 23. The method of claim 1 wherein the firstpharmaceutical composition comprises a drug that inhibits at least oneof a T-reg cell, a myeloid derived suppressor cell, and a M2 macrophage.24. The method of claim 23 wherein the drug is selected from the groupconsisting of cisplatin, gemcitabine, 5-fluorouracil, cyclophosphamide,doxorubicin, temozolomide, docetaxel, paclitaxel, trabectedin, andRP-182 (see U.S. Pat. No. 9,492,499).
 25. The method of claim 1 whereinthe first pharmaceutical composition comprises a vascular permeabilityenhancer.
 26. The method of claim 25 wherein the first vascularpermeability enhancer comprises at least a portion of IL2.
 27. Themethod of claim 1 wherein the second pharmaceutical compositioncomprises a recombinant bacterial vaccine, a recombinant viral vaccine,or a recombinant yeast vaccine.
 28. The method of claim 27 wherein therecombinant bacterial vaccine, the recombinant viral vaccine, or therecombinant yeast vaccine is genetically engineered to express at leastone of a tumor associated antigen and a patient and tumor specificneoepitope.
 29. The method of claim 28 wherein the tumor associatedantigen is selected from the group consisting of MUC1, CEA, HER2,Brachyury, and an oncogenic Ras mutant protein.
 30. The method of claim1 wherein the second pharmaceutical composition comprises a naturalkiller cell.
 31. The method of claim 30 wherein the natural killer cellis an aNK cell, a haNK cell, or a taNK cell.
 32. The method of claim 1wherein the second pharmaceutical composition comprises an immunestimulatory cytokine.
 33. The method of claim 32 wherein the immunestimulatory cytokine is selected from the group consisting of IL-2,IL-7, IL-15, IL-17, IL-21, and an IL-15 superagonist.
 34. The method ofclaim 1 wherein the third pharmaceutical composition comprises at leastone of a checkpoint inhibitor, an immune stimulatory cytokine, arecombinant bacterial vaccine, a recombinant viral vaccine, and arecombinant yeast vaccine.
 35. The method of claim 34 wherein thecheckpoint inhibitor is a PD-1 inhibitor or a CTLA4 inhibitor, andwherein the immune stimulatory cytokine is selected from the groupconsisting of IL-2, IL-7, IL-15, IL-17, IL-21, and an IL-15superagonist.
 36. The method of claim 1 further comprising a step ofadministering low dose radiation to the tumor. 37-84. (canceled)